AU2013276219A1 - Conjugates of biologically active molecules to functionalized polymers - Google Patents

Conjugates of biologically active molecules to functionalized polymers

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AU2013276219A1
AU2013276219A1 AU2013276219A AU2013276219A AU2013276219A1 AU 2013276219 A1 AU2013276219 A1 AU 2013276219A1 AU 2013276219 A AU2013276219 A AU 2013276219A AU 2013276219 A AU2013276219 A AU 2013276219A AU 2013276219 A1 AU2013276219 A1 AU 2013276219A1
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derivative
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alkyleneoxyalkylene
alkylene
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Marek Kwiatkowski
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QuiaPEG Pharmaceuticals AB
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QUIAPEG AB
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    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
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    • C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
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Abstract

This document relates to conjugates of a biologically active molecule or a derivative thereof and functionalized (e.g., mono- or bi-functional) polymers (e.g., polyethylene glycol and related polymers) as well as methods and materials for making and using such conjugates.

Description

WO 2013/186632 PCT/IB2013/001885 Conjugates of Biologically Active Molecules to Functionalized Polymers CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Serial Nos. 61/658,827, filed June 12, 2012; 61/785,996, filed March 14, 2013; 61/658,839, filed June 12, 2012; 61/786,121, filed March 14, 2013; 61/658,835, filed June 12, 2012; 61/786,162, filed March 14, 2013; 61/658,836, filed June 12, 2012; 61/786,237, filed March 14, 2013; 61/658,850, filed June 12, 2012; 61/786,221, filed March 14, 2013; 61/658,853, filed June 12, 2012; 61/786,265, filed March 14, 2013; 61/658,856, filed June 12, 2012; and 61/786,287, filed March 14, 2013; all of which are incorporated by reference in their entireties. TECHNICAL FIELD This document relates to conjugates of a biologically active molecule to a functionalized (e.g., mono- or bi-functional) polymers (e.g., polyethylene glycol and related polymers) as well as methods and materials for making and using such conjugates. BACKGROUND Pharmacokinetic and immune stimulating properties of proteins and synthetic drugs may be controlled by their conjugation to certain polymers. For example, polyethylene glycol (PEG) can be conjugated to proteins to achieve this effect (Fee and Van Alstine, Chemical Engineering Science, 61:924-934 (2006)). Such conjugation can take place if the relatively non-reactive hydroxyl groups present in PEG molecules are substituted by other, more reactive moieties (Jagur-Grudzinski, Reactive & Functional Polymers, 39:99-138 (1999)). A standard, linear PEG molecule is chemically a diol, which could suggest that the process of PEG derivatization and purification of products should be trivial. However, the polymeric nature of this diol, together with its amphiphilic properties can make these manipulations difficult. In some cases, the typical laboratory process for separation of difficult reaction mixtures, silica gel-based flash column chromatography, can fail for PEG with molecular weight higher than 1000. Neither crystallization nor precipitation appear adequate to achieve separation of PEG-containing materials, even if these methods can be used for efficient removal of other, contaminating 1 WO 2013/186632 PCT/IB2013/001885 substances with low molecular weight. Most reaction mixtures containing modified PEG molecules lack a reliable analytical method to control or to prove their composition. Polymers with functions that influence only minimally the hydrophobic properties of the polymer can be difficult to analyze by chromatography. The same applies for polymers with functions carrying only a minimal charge. This also applies for preparative chromatographic separation of charged polymers as described elsewhere for the separation of mono- and di-carboxyl modified PEG molecules (Drioli et al., Reactive & Functional Polymers, 48:119-128 (2001)). Confirmation of results of the synthesis based on NMR can be useless, as long as one is not sure about the purity of the product, and this is typically only obtained by chromatographic methods. This unusual conclusion comes from observations that an equimolar mixture of non derivatized polymer and bis-derivatized polymer will produce an NMR pattern identical to the pure mono-derivatized polymer. Mass spectrometry can be complicated since most PEG exists not in the form of a single component, but is rather a Gaussian population of different polymer lengths, centered on its average molecular weight. Thus, even if all distinct components of the same type should have their mass increased by the same factor, the presence of unreacted and bis-modified material can obscure the picture of the analysis. The literature discusses this problem only sporadically, and often nothing is mentioned about analysis of the product or its purification. Many authors make the impression that the process that they describe is ongoing with quantitative yield, and thus the quality of the product does not need to be analyzed or questioned. This non-scientific approach can be frequently encountered in the chemistry of PEG. There are many examples in the literature presenting synthetic procedures with four to five consecutive steps without a single analysis of the product at any of these steps, without any attempts to purifying the product, and assuming 100 percent purity at the end of the process. It is, therefore, not strange that researchers after closer testing question these products and their purity (Ananda et al., Anal. Biochem., 374, 231-242 (2008)). A commonly accepted escape from the problem of selective modification is to work with a polymer that has one end blocked from the beginning by a stable chemical group, most often a methoxy group (mPEG). In theory, this blockage converts a PEG molecule to a monofunctional compound, and as such, it could be fully converted to the second derivatized form by increasing the amount of derivatizing reagent and/or time for reaction. Unfortunately, many of reactions commonly applied for derivatization of PEG 2 WO 2013/186632 PCT/IB2013/001885 are sluggish and only seldom go to completion. On the other hand, mPEG preparations contain significant percentages of PEG diol component. Moreover, the amount of this contamination increases with the length of mPEG, and this contamination can be hard to avoid. Consequently, derivatization will also result in formation of symmetrical, bis-derivatizated PEG, and its presence in the conjugating mixture results in formation of cross-linked products with unknown pharmacologic properties or a possible loss of protein activity. Therefore, pure, monofunctional polymers are usually preferred for protein modification, but one should be aware that purification of mPEG from its diol PEG contamination is practically impossible. Nearly all of existing reactions, used today for derivatization of PEG, belong either to the alkylation-based or the acylation-based category. In the first case, the alkoxy anion, generated from PEG, is reacting with incoming electrophilic modifying reagent. Eventually, the activated PEG, subjected with a good leaving group, is itself an object of a nucleophilic attack. To this category belong processes resulting in thiolation, amination, azidation, and introduction of a carboxyl or an aldehyde group. Modified PEG's of this category will have their functional group connected directly to the PEG terminal carbon atom or these groups will be linked via an ether bond, a thioether bond, or a secondary amino group. The second category, acylation, is based on a nucleophilic reaction of PEG's hydroxyl, (or another group present in a modified PEG - often an amino group), on an incoming acylating reagent. In many cases, this first acylation is followed by a second acylation that actually introduces the modification of interest to the PEG molecule. Functional groups incorporated by this method can be linked to the rest of PEG by an amido, a carbamido, urethane, thiourethane, or a simple ester group. These linking groups and the chemistry behind them belong to the very traditional methods of combining two chemical identities. Polyethylene glycols (PEG) coupled to phosphoramidites are used for direct coupling of PEG molecules to synthetic nucleic acids. One example is 4,4'-dimethoxytrityl polyethyleneglycol-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. In these compounds, the phosphoramidite group is the part of the reactive functionality for linking the compound to a synthetic nucleic acid. It is designed to work in a completely water-free environment: In the presence of water, the phosphoramidite group can decompose instantaneously, making such PEG phosphoramidites inappropriate for conjugation to biological material in water-containing or 3 WO 2013/186632 PCT/IB2013/001885 aqueous solution. In particular, these PEG phosphoramidites can be inappropriate for conjugation to biological substances which are not soluble, stable or sufficiently reactive in non aqueous media. Furthermore, already mildly acidic biological substances can decompose these PEG phosphoramidites. Finally, these PEG phosphoramidites contain a labile protecting group adjacent to the phosphorous atom which is specially designed to convert the intermediate phosphotriester to a phosphodiester. Phosphodiesters can be readily degraded enzymatically in vivo. SUMMARY This document provides conjugates of a biologically active molecule (e.g., TNF inhibitors, insulin, omalizumab, clotting factors, polypeptides that boost red or whit blood cell production, an antibody), or derivatives thereof to functionalized (e.g., mono- or bi-functional) polymers (e.g., polyethylene glycol and related polymers) as well as methods and materials for making and using such conjugates. The conjugates described herein can have an altered pharmacokinetic and pharmacodynamic profile, including, for example, one or more of reduced dosage frequency, extended circulation time, and reduced antigenic properties. The functionalized polymers include one or more linking groups selected from a phosphotriester, a phosphoramidate, a thiophosphotriester, and a thiophosphoramidate. For example, a biologically active molecule or a derivative thereof can be conjugated to a PEG polymer having at least one functional group at one terminus covalently bound to the rest of the polymer via a phosphotriester or phosphoramidate linking group. A biologically active molecule or a derivative thereof can be conjugated to a functionalized polymer provided herein that includes different linking groups at each of its termini. Also provided herein is a biologically active molecule or a derivative thereof conjugated to a functionalized polymer in which at least one terminus is modified with a blocking group (e.g., methoxy group) and at least another terminus is functionalized with a linking group as described herein. As described herein, preparations of a functionalized polymer having one or more functional groups linked as described herein can be obtained in a manner where greater than 50 percent by weight (e.g., greater than 75 percent, greater than 80 percent, greater than 90 percent, greater than 95 percent, greater than 98 percent, and greater than 99 percent by weight) of the 4 WO 2013/186632 PCT/IB2013/001885 preparation is the desired functionalized polymer free from contaminants and can be used to conjugate to a biologically active molecule or a derivative thereof. As a person of ordinary skill in the art would understand, a functionalized polymer preparation includes a Gaussian population of different polymer lengths centered on an average molecular weight, and such a functionalized population would not be considered contaminating. For example, a mono-functionalized PEG population can be separated nearly quantitatively from contaminants of PEG functionalization (e.g., unreacted PEG and poly-functionalized PEG populations). The separation of a functionalized polymer provided herein can be facilitated through the use of a removable hydrophobic separation handle (e.g., a substituted or unsubstituted trityl group) which upon removal allows for preparative isolation of product (e.g., pure product), free or substantially free from unreacted polymer and poly-functionalized polymer. Having the ability to isolate functionalized polymers in high purity can allow chemists to more easily control subsequent reactions or the purity of the downstream products, e.g., conjugates to a biologically active molecule or a derivative thereof. In some cases, having the ability to introduce all functional groups through a unified process using similar, mild reagents and reaction conditions can allow for the production of a functionalized polymer through a fast and nearly quantitative reaction. The ability to isolate functionalized polymers quantitatively can allow chemists to more easily control subsequent reactions or the purity of conjugates of a biologically active molecule such as a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, or an antibody, coupled to a functionalized polymer provided herein. A linking group, in addition to linking the functional group or separation handle, can act as a linker between the polymer and a biologically active molecule or derivative thereof. For instance, a functionalized polymer of high purity can be coupled to a biologically active molecule or derivative thereof. In such conjugates, the linker can form covalent bonds to the polymer and a biologically active molecule or derivative thereof. The coupling can take place in an aqueous reaction medium. Furthermore, the linking groups provided herein are generally resistant to chemical and enzymatic degradation, providing for stable storage and increased safety and efficacy in vivo. 5 WO 2013/186632 PCT/IB2013/001885 In one aspect, this document features a method of making a biologically active molecule conjugate. The method includes reacting a biologically active molecule or a derivative thereof with a preparation comprising a water-soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (1) under conditions suitable for group M to react with a biologically active molecule or the derivative thereof: E R1 A -0-P-Z 1 -L -M-R Z2 K I G or a salt thereof, wherein A is the point of covalent bonding to the terminus of the polymer backbone; E is 0 or S; K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic separation handle; Z' and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; M is a protected group that when deprotected is reactive with a biologically active molecule or derivative thereof or is a group reactive with a biologically active molecule or derivative thereof; R is absent or selected from the group consisting of: hydrogen, a protecting group, a hydrophobic separation handle, or an activating group; R 1 is absent or a hydrophobic separation handle; wherein when M is a protected group that when deprotected is reactive with a biologically active molecule or derivative thereof, then R is a protecting group or a hydrophobic separation handle; wherein when M is a group reactive with a biologically active molecule or derivative thereof, R is absent, hydrogen, or an activating group; and wherein only one of R, R 1 , and G can be a hydrophobic separation handle. In some embodiments, the polymer backbone has from 2 to 100 termini. In some embodiments, only one termini of the polymer backbone is covalently bonded to the structure of formula (1). 6 WO 2013/186632 PCT/IB2013/001885 In some embodiments, only one termini of the polymer backbone is covalently bonded. In some embodiments, the polymer backbone has two termini. In some embodiments, only one termini of the polymer backbone is covalently bonded to the structure of formula (1). In some embodiments, both termini of the polymer backbone are covalently bonded to the structure of formula (1). A polymer backbone can be selected from the group consisting of poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol), polyoxazoline, and copolymers. For example, a polymer backbone can be poly(ethylene glycol). In some cases, a poly(ethylene glycol) has an average molecular weight from about 500 Da to about 100,000 Da. In some embodiments, one of Z' and Z 2 is NH and the other is 0. In some embodiments, Z' is 0 and Z 2 is NH. In some embodiments, Z' is NH and Z 2 is 0. In some embodiments, both Zi and Z2 are 0. The group reactive with a biologically active molecule or the derivative can be selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, and iodoacetamide. In some embodiments, K is selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, G is a substituted or unsubstituted trityloxy. In some embodiments, L is a substituted or unsubstituted C1-C 1 2 alkylene. In some embodiments, R is selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal. In some embodiments, a group reactive with a biologically active molecule or the derivative is carboxyl and R is absent or selected from the group consisting of N hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl. In some embodiments, the preparation includes at least 50% by weight (e.g., at least 60%, 70%, 80%, 90%, 95%, or 98% by weight) of the water-soluble, non-peptidic, and non 7 WO 2013/186632 PCT/IB2013/001885 nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (1). In some embodiments, the method further can include (i) removing the hydrophobic separation handle(s) from the structure of formula (1) and (ii) optionally reacting the compound obtained in step (i) with an activating agent before reacting with a biologically active molecule or the derivative. In some embodiments, the method further can include removing the hydrophobic separation handle(s) from the structure of formula (1) after reacting with a biologically active molecule or the derivative. This document also features a conjugate, or a pharmaceutically acceptable salt thereof, that includes a water-soluble, non-peptidic, and non-nucleotidic polymer backbone as in a structure of formula (9): E R1 1 1 1 | A-O-P-Z- L 2 -B Z2 K G or a salt thereof, wherein A is the point of covalent bonding to the terminus of the polymer backbone; E is o or S; K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic separation handle; Z' and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; R 1 is absent or a hydrophobic separation handle, wherein only one of R 1 and G can be a hydrophobic separation handle; L 2 is a covalent linking moiety between L on the polymer backbone and B; and B is a biologically active molecule or a derivative thereof. 8 WO 2013/186632 PCT/IB2013/001885 This document also features a method of making a biologically active molecule conjugate. The method includes reacting a biologically active molecule or a derivative thereof with a preparation that includes a compound of formula (2) under conditions suitable for group M to react with a biologically active molecule or the derivative thereof: El E | |1 1 1
R
1
-M
1
-L-Z
3 -P-O polymer O-P-Z 1 -L-M-R I I z4 Z2 Gi-K1 K-G (2) or a salt form thereof, wherein polymer is a linear, water-soluble, non-peptidic, and non nucleotidic polymer backbone, wherein each linking group is bonded at a different terminus of the polymer; E and El are independently 0 or S; K and K 1 are independently selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G and G1 are independently absent or are selected from the group consisting of: alkoxy and a hydrophobic separation handle; each pair of Z' and Z 2 and Z 3 and Z 4 are independently selected from 0 and NH, wherein only one of each pair of Z' and Z 2 and Z 3 and Z 4 can be NH; L and L' are independently selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; M and M 1 are independently selected from a protected group that when deprotected is reactive with a biologically active molecule or the derivative or a group reactive with a biologically active molecule or the derivative, wherein M and M 1 are different; and R and RI are independently absent, hydrogen, a protecting group, or an activating group; wherein when M is a protected group that when deprotected is reactive with a biologically active molecule or the derivative, then R is a protecting group or a hydrophobic separation handle; wherein when M is a group reactive with a biologically active molecule or the derivative, R is absent, hydrogen, or an activating group; wherein when M 1 is a protected group that when deprotected is reactive with a biologically active molecule or the derivative, then R 1 is a protecting group or a 9 WO 2013/186632 PCT/IB2013/001885 hydrophobic separation handle; and wherein when M 1 is a group reactive with a biologically active molecule or the derivative, R1 is absent, hydrogen, or an activating group. In some embodiments, one of Z' and Z 2 is NH and the other is 0. In some embodiments, Z' is 0 and Z 2 is NH. In some embodiments, Z' is NH and Z 2 is 0. In some embodiments, both ZI and Z 2 are 0. In some embodiments, one of Z 3 and Z 4 is NH and the other is 0. In some embodiments, Z3 is 0 and Z4 is NH. In some embodiments, Z 3 is NH and Z 4 is 0. In some embodiments, both Z 3 and Z 4 are 0. In some embodiments, the group reactive with a biologically active molecule or the derivative thereof is selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, and iodoacetamide. In some embodiments, K and K' are independently selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, G and G 1 is independently seleceted from a substituted or unsubstituted trityloxy. In some embodiments, L and L' is independently a substituted or unsubstituted CI-C 12 alkylene. In some embodiments, R or R 1 is independently selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and combinations of thereof. In some embodiments, the polymer is poly(ethylene glycol). For example, a poly(ethylene glycol) having an average molecular weight from about 500 Da to about 100,000 Da. In some embodiments, the preparation includes at least 50% by weight (e.g., at least 60%, 70%, 80%, 90%, 95%, or 98% by weight) of the water-soluble, non-peptidic, and non nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (2). 10 WO 2013/186632 PCT/IB2013/001885 In some embodiments, the method further can include (i) removing the hydrophobic separation handle(s) from the structure of formula (1) and (ii) optionally reacting the compound obtained in step (i) with an activating agent before reacting with a biologically active molecule or the derivative. This document also features a conjugate, or a pharmaceutically acceptable salt thereof, including a structure of formula (10): El E
B
1 - L 3
-L
1 -- Z 3 -- P - O- polymer- - P- Z 1 -- L L 2 -B z4 Z2 G1-K1 K--G (10) or a salt form thereof, wherein: polymer is a linear, water-soluble, non-peptidic, and non nucleotidic polymer backbone, wherein each linking group is bonded at a different terminus of the polymer; E and El are independently 0 or S; K and K 1 are independently selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G and G1 are independently absent or are selected from the group consisting of: alkoxy and a hydrophobic separation handle; each pair of Z' and Z 2 and Z 3 and Z 4 are independently selected from 0 and NH, wherein only one of each pair of Z' and Z 2 and Z 3 and Z 4 can be NH; L and L' are independently selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; L 2 is a covalent linking moiety between L on the polymer backbone and B; L3 is a covalent linking moiety between L on the polymer backbone and B 1 ; and B and B are independently a biologically active molecule or a derivative of a biologically active molecule (e.g., a TNF inhibitor, a derivative of a TNF inhibitor, insulin, a derivative of insulin, omalizumab, a derivative of omalizumab, a clotting factor, a derivative of a clotting factor, a polypeptide that boosts red or white cell production, a derivative of a polypeptide that boosts red or white cell production, an antibody, or a derivative of an antibody), a drug, a detectable group, a separation moiety, wherein at least one of B and B 1 is a biologically active molecule or a 11 WO 2013/186632 PCT/IB2013/001885 derivative of a biologically active molecule. For example, B and B 1 each can be selected from the group consisting of: a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, and an antibody. This document also features a method of making a biologically active molecule conjugate. The method includes reacting a biologically active molecule or a derivative thereof with a preparation that includes a compound of formula (3): E 2M2-polymer O-P-Z 1 -L-M-R Z2 K- G (3) or a salt form thereof, wherein: polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer backbone, wherein M 2 and the phosphonate-derived functional group are bonded at a different terminus of the polymer; E and El are independently 0 or S; K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic separation handle; Z' and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; M is selected from a protected group that when deprotected is reactive with a biologically active molecule or the derivative or a group reactive with a biologically active molecule or the derivative; M 2 is selected from 0, S or NH; and R is absent, a protecting group, a hydrophobic separation handle, or an activating group; R 2 is hydrogen or a protecting group; wherein when M is a protected group that when deprotected is reactive with a biologically active molecule or the derivative, then R is a protecting group or a hydrophobic separation handle; and wherein when M is a group reactive with a biologically active molecule or the derivative, R is absent, hydrogen, or an activating group. 12 WO 2013/186632 PCT/IB2013/001885 In some embodiments, one of Z' and Z 2 is NH and the other is 0. In some embodiments, Z' is 0 and Z 2 is NH. In some embodiments, Z' is NH and Z 2 is 0. In some embodiments, both Zi and Z2 are 0. A group reactive with a biologically active molecule or the derivative can be selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, and iodoacetamide. In some embodiments, K is selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, G is a substituted or unsubstituted trityloxy. For example, G can be a monoalkoxy substituted trityloxy group or a dialkoxy substituted trityloxy group. In some embodiments, R 2 is absent or selected from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl, and methyl. In some embodiments, the polymer is poly(ethylene glycol). In some embodiments, the preparation includes at least 50% by weight (e.g., at least 60%, 70%, 80%, 90%, 95%, or 98% by weight) of the water-soluble, non-peptidic, and non nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (3). In some embodiments, the method further can include (i) removing the hydrophobic separation handle(s) from the structure of formula (1) and (ii) optionally reacting the compound obtained in step (i) with an activating agent before reacting with a biologically active molecule or the derivative. In some embodiments, the method further can include removing the hydrophobic separation handle(s) from the structure of formula (1) after reacting with a biologically active molecule or the derivative. This document also features a conjugate, or a pharmaceutically acceptable salt thereof, that includes a compound of formula (11): 13 WO 2013/186632 PCT/IB2013/001885 E Bl- L 4 polymer-O- P- Z - L Z2 B K- G (11) or a salt form thereof, wherein: polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer backbone, wherein M 2 and the phosphonate-derived functional group are bonded at a different terminus of the polymer; E and El are independently 0 or S; K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic separation handle; Z' and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; L2 is a covalent linking moiety between L on the polymer backbone and B; L4 is a covalent linking moiety between L on the polymer backbone and B 1 ; and B and B 1 are independently a biologically active molecule or a derivative of a biologically active molecule (e.g., a TNF inhibitor, a derivative of a TNF inhibitor, insulin, a derivative of insulin, omalizumab, a derivative of omalizumab, a clotting factor, a derivative of a clotting factor, a polypeptide that boosts red or white cell production, a derivative of a polypeptide that boosts red or white cell production, an antibody, or a derivative of an antibody), a biologic other than the biologically active molecule, a drug, a detectable group, a separation moiety, wherein at least one of B and B 1 is a biologically active molecule or a derivative of a biologically active molecule. For example, B and B 1 each can be selected from the group consisting of: a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, an antibody, This document also features a method of preparing a compound that includes a water soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (9): 14 WO 2013/186632 PCT/IB2013/001885 E R1 A -O-P-Z -L-L 2 -- B Z2 '*Y (9) or a salt thereof, wherein: A is the point of covalent bonding to the terminus of the polymer backbone; E is o or S; Y represents an optionally substituted residue selected from alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; Z' and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; L2 is a covalent linking moiety between L on the polymer backbone and B; and B is a biologically active molecule or a derivative thereof. The method includes (a) providing a composition comprising a compound of formula (8): E R1 1 1 1 | A-O--P--Z1--L-M--R Z2 Y(8) wherein: A is the point of covalent bonding to the terminus of the polymer backbone; E is o or S; Y represents an optionally substituted residue selected from alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; Z' and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; M is a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof; R is a hydrophobic separation handle;
R
1 is absent or a hydrophobic separation handle; and (b) removing the hydrophobic separation handle(s); (c) optionally reacting the compound obtained in step (b) with an activating agent; and 15 WO 2013/186632 PCT/IB2013/001885 (d) reacting the compound obtained in step (b), or, optionally in step (c), with a biologically active molecule or a derivative thereof. In some embodiments, one of Z' and Z 2 is NH and the other is 0. In some embodiments, Z' is 0 and Z 2 is NH. In some embodiments, Z' is NH and Z 2 is 0. In some embodiments, both Z' and Z2 are 0. The protected group M can be, when deprotected, selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, and iodoacetamide. In some embodiments, R is selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal. In some embodiments, the polymer backbone is selected from the group consisting of poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol), polyoxazoline, and copolymers. For example, the polymer backbone can be poly(ethylene glycol). In some embodiments, reaction step (d) is carried out in the presence of water or a protic solvent. In some embodiments, the compound of formula (8) is essentially pure. In some embodiments, the compound of formula (8) comprises at least 98% by weight of the composition. In one aspect, this document features a composition that includes any of the conjugates described herein and a pharmaceutically acceptable excipient. The polymer backbone of the conjugate can be poly(ethylene glycol). In yet another aspect, this document features a method of treating a patient diagnosed with a disease selected from the group consisting of: an inflammatory disease; diabetes; asthma; hemophilia, factor VII deficiency, or other clotting disorder; The method includes administering to the patient an effective amount of any of the conjugates described herein. The polymer backbone of the conjugate can be poly(ethylene glycol). This disclosure utilizes phosphoramidites as reagents interacting with polymers to form a phosphotriester-type of linker between a polymer and linking group. A similar process is used commonly in the chemistry of nucleic acids. Formation of a phosphotriester bond, in the 16 WO 2013/186632 PCT/IB2013/001885 chemistry of nucleic acids, is often followed by its partial hydrolysis (deprotection) to the phosphodiester bond, because phosphodiester bonds are naturally occurring. The present disclosure provides an uncommon and unnatural phosphotriester linkage which is enzymatically resistant, offering a stable linker between a polymer and a biologically active molecule such as a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, an antibody, or a derivative thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF THE DRAWINGS Figure 1 shows SEQ ID NO.1, the amino acid sequence omalizumab (XOLAIR T M ). Figure 2 provides a photograph of a SDS electrophoresis gel of the product of the conjugation of omalizumab to a PEG reagent as provided herein stained with Coomassie blue. Figure 3 provides a photograph of a SDS electrophoresis gel of the product of the conjugation of omalizumab to a PEG reagent as provided herein stained with barium chloride and iodine. Figure 4 provides a photograph of a SDS electrophoresis gel of the product of the conjugation of omalizumab to a PEG reagent as provided herein stained with Coomassie blue. Figure 5 provides a photograph of a SDS electrophoresis gel of the product of the conjugation of omalizumab to a PEG reagent as provided herein stained with Coomassie blue. 17 WO 2013/186632 PCT/IB2013/001885 Figure 6 shows HPLC Gel Filtration analysis of free omalizumab and reaction mixture obtained after pegylation of omalizumab. Figure 7 shows SEQ ID NO.2, the amino acid sequence of insulin. Figure 8 illustrates the HPLC Gel Filtration chromatogram of lysozyme (dashed line) and the lysozyme pegylation reaction product (solid line). Figure 9 illustrates the HPLC Gel Filtration chromatogram of insulin (dashed line) and insulin pegylation reaction mixture (solid line). Figure 10 shows SEQ ID NO.3, the amino acid sequence etanercept (ENBREL@). Figure 11 provides a photograph of a SDS electrophoresis gel of the product of the conjugation of entanercept to a PEG reagent as provided herein stained with Coomassie blue. Figure 12 illustrates the HPLC Gel Filtration chromatogram of etanercept (dashed line) and the etanercept pegylation reaction product (solid line). Figure 13 shows rhTNF-alpha concentrations following over-night incubation at 4-8' C with various concentrations of etanercept (non-PEGylated), PEG20-etanercept (5 eq), or PEG20 etanercept (10 eq). A fixed concentration of 2.9 ng/ml rhTNF-alpha was used in all incubations. PEGylation was conducted in 5 time molar excess (5 eq) or 10 time molar excess (10 eq) of PEG20 compared etanercept. IC 50 is expressed as gram etanercept per ml. Figure 14 illustrates the dissociation over time of 125 1-omalizumab bound to human IgE with or without unlabeled omalizumab or PEGylated omalizumab. DETAILED DESCRIPTION This document provides conjugates of a biologically active molecule (e.g., a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, or an antibody) or a derivative thereof and functionalized (e.g., mono- or bi functional) polymers (e.g., polyethylene glycol and related polymers) as well as methods and materials for making and using such conjugates. Non-limiting examples of a biologically active molecule include a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, and an antibody. 18 WO 2013/186632 PCT/IB2013/001885 As used herein, the term "TNF inhibitor" includes antibodies or fusion proteins that bind to TNF alpha. Non-limiting examples of TNF inhibitors include etanercept (Enbrel@, sold by Amgen and Pfizer); infliximab (Remicade@, sold by Janssen Biotech, Inc.); adalimumab (Humira@, sold by Abbott Laboratories); certolizumab pegol (Cimzia@); and Golimumab (Simponi@, sold by Janssen Biotech, Inc.). Etanercept (Enbrel@) is a fusion protein of human soluble TNF receptor 2 to the Fc component of human IgG 1 . It is a TNF inhibitor that binds to TNF alpha and is used to treat inflammatory diseases e.g., rheumatoid arthritis, plaque psoriasis, psoriatic arthritis, juvenile idiopathic arthritis (JIA), and ankylosing spondylitis (AS)). Infliximab (Remicade@) is a chimeric mouse-human monoclonal antibody that specifically binds TNF alpha. It is used for the treatment of psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, and ulcerative colitis. Adalimumab (Humira@) is a fully human monoclonal antibody that binds TNF alpha and is used for the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, moderate to severe chronic psoriasis, and juvenile idiopathic arthritis. Certolizumab pegol (Cimzia@) is a pegylated fragment Fab' of humanized TNF inhibitor monoclonal antibody. Golimumab (Simponi@) is a human monoclonal antibody that targets TNF alpha, and is used to treat severely active rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. As used herein, the term "insulin" includes human and other mammalian insulin polypeptides as well as insulin analogs that have one or more amino acid substitutions. Human insulin is composed of A and B polypeptide chains bound together by disulfide bonds, where the A chain is 21 amino acids in length and the B chain is 30 amino acids in length, 51 amino acids in total. Insulin and analogs can be recombinantly produced (e.g., in a non-pathogenic organism). Non-limiting examples of suitable insulin analogs include insulin lispro (HUMALOG, Eli Lilly), insulin aspart (NovoLog/NovoRapid, Novo Nordisk), insulin detemir (Levemir@, Novo Nordisk), Insulin glargine (Lantus@, Sanofi-Aventis), and insulin glulisine (Apidra@, Sanofi-Aventis). See Werner and Chantelau, Diabetol Metab Syndr. 3: 13 (2011) for a comparison of the bioactivity of human insulin and insulin analogs. 19 WO 2013/186632 PCT/IB2013/001885 Insulin lispro is a rapid-acting human insulin analog used to lower blood glucose levels, and contains a substitution of lysine for proline at position B28, and a substitution of proline for lysine at position B29. Insulin aspart is a rapid-acting human insulin analog used to lower blood glucose levels, and contains a substitution of an aspartic acid for proline at position B28. Insulin detemir is a long-acting insulin analog used for maintaining the basal level of insulin. Insulin determir contains myristic acid is bound to the lysine at position B29. Insulin glargine is a long-acting insulin analog used for lower blood glucose, and contains a substitution of a glycine for asparagine at position A21 and contains two arginines added to the C-terminus of the B-chain. Insulin glulisine is a rapid acting human insulin analog used to lower blood glucose levels, and contains a substitution of a lysine for asparagine at position B3 and the substitution of a glutamic acid for lysine at position B29. Omalizumab (Xolair®, Novartis Pharmaceuticals, East Hanover, NJ; Genentech Inc., South San Francisco, CA) is a recombinant humanized monoclonal antibody (rhuMAb-E25) for treatment of moderate to severe persistent asthma. Omalizumab inhibits the binding of IgE to the high-affinity IgE receptor FcRI by binding to an epitope on IgE that overlaps with the site to which FcRI binds. Consequently, circulating free IgE is reduced, IgE is prevented from attaching to mast cells and basophils, and FccRI receptor expression is down-regulated. See, e.g., Sandstrom, JAsthma Allergy, 2: 49-62 (2009). As used herein, the term "clotting factor" includes human or other mammalian clotting factors such as factor VII and activated factor VII (factor VIa), factor VIII and activated factor VIII (factor VIlla), factor IX and activated factor IX (factor IXa), antithrombin, human fibrinogen / human thrombin, and protein C and activated protein C (APC). Such clotting factors can be recombinantly produced. Factor VII functions in the initial stage of blood clotting and is a key element in forming blood clots. The inactive precursor has low enzyme activity that is increased by proteolytic cleavage to form factor VIa. This activation can be catalyzed by factor Xa as well as by VIIa tissue factor, an integral membrane protein found in a number of cell types. See Fiore, et al., J. Biol. Chem., 269:143-149 (1994). Factor VIa can activate blood clotting factors IX and X. 20 WO 2013/186632 PCT/IB2013/001885 Eptacog alfa (activated) (sold by Novo Nordisk) can be used to replace missing factor VII in patients with factor VII deficiency and also can be used in haemophilia patients who have developed inhibitors to factor VIII or IX. Factor VIII is activated by thrombin (Factor Ila), and then can interact with Factor IXa in the coagulation cascade. It is a cofactor to Factor IXa in the activation of Factor X, which, in turn, with its cofactor Factor Va, activates more thrombin. Thrombin cleaves fibrinogen into fibrin which polymerizes and crosslinks (using Factor XIII) into a blood clot. Factor VIII deficiency results in hemophilia A and Factor IX deficiency causes hemophilia B. Moroctocog alfa (glycoprotein (trade name ReFacto@, Xyntha@) are commercially available for Factor VIII replacement therapy. Factor VIII replacement therapy is limited due to development of high-titer inhibitory factor VIII antibodies in some patients. Nonacog alfa (Benefix@) is a human recombinant factor IX product used in treating hemophilia B patients. Antithrombin is a small glycoprotein that inactivates several enzymes of coagulation system. Its activity enhanced by heparin. Antithrombin alfa (e.g., ATryn@) is commercially available. Protein C is activated by thrombin in the presence of thrombomodulin, an integral membrane protein of endothelial cells. Esmon, et al., J. Biol. Chem., 257:859-864 (1982). Activated protein C (APC) degrades factors Va and VIIIa in combination with its cofactor, protein S. Resistance to APC is the most common form of inherited thrombosis disease. Dahlback, Blood, 85:607-614 (1995). As used herein, the term "polypeptide that boosts red blood cell production" includes human erythropoetin (e.g., epoetin alpha (Epogen@, Amgen), epoetin beta (NeoRecormon@, Roche), epoetin theta (Eporatio@), or epoetin zeta) and other mammalian erythropoetin polypeptides as well as analogs that have one or more amino acid substitutions (e.g., darbepoetin alpha (Aranesp@, Amgen)) or other chemical modifications. As used herein, the term "polypeptide that boosts white blood cell production" includes granulocyte colony-stimulating factor (G-CSF) analogs such as filgrastim (e.g., Neupogen@), which is identical to human G CSF except for the addition of an N-terminal methionine necessary for expression in E coli. Figrastim is nonglycosylated. Such polypeptides can be recombinantly produced. 21 WO 2013/186632 PCT/IB2013/001885 Epoetin alpha, beta, theta, and zeta are structurally similar and have the same polypeptide receptor binding sites. However, there are subtle differences in the glycosylation patterns of different types of epoetins. See Sergei et al., Arch Drug Inf, 4(3): 33-41 (2011); and Storring et al., Br JHaematol. 100(1):79-89 (1998). Darbepoetin is a 165-amino acid protein that differs from recombinant human erythropoietin in containing 5 N-linked oligosaccharide chains, whereas recombinant human erythropoietin contains 3 chains. The two additional N glycosylation sites result from amino acid substitutions in the erythropoietin peptide backbone. The additional carbohydrate chains increase the approximate molecular weight of the glycoprotein from 30,000 to 37,000 daltons. Non-limiting examples of antibodies that can be conjugated to a functionalized polymer include, abatacept (Orencia@), alemtuzumab (marketed as Campath, MabCampath or Campath 1H); basiliximab (Simulect@), belimumab (Benlysta@), besilesomab (Scintimun@), bevacizumab (Avastin@), canakinumab (Ilaris@), catumaxomab (Removab@), cetuximab (Erbitux@), denosumab (Prolia@, Xgeva@), eculizumab (Soliris@), ipilimumab (also known as MDX-010 or MDX-101, Yervoy@), natalizumab (Tysabri@), ofatumumab (Arzerra@), palivizumab (Synagis@), panitumumab (Vectibix@), ranibizumab (Lucentis@), rituximab (Rituxan@, MabThera@), tocilizumab (Actemra@, RoActemra@), trastuzumab (Herceptin@), and ustekinumab (Stelara@). The antibody also can be an antibody drug conjugate (ADC). An ADC can include, for example, an antibody such as a monoclonal antibody, or antibody fragment such as a single chain variable fragment, linked to a drug (e.g., a cytotoxic drug). In some embodiments, the antibody or fragment and drug can be linked via a cleavable linker (e.g,. a disulfide-based linker or peptide linker). In some embodiments, the antibody or fragment and drug can be linked via a noncleavable linker. For example, this document provides an antibody or a derivative thereof such as abatacept (Orencia@), alemtuzumab (marketed as Campath, MabCampath or Campath-1H); basiliximab (Simulect@), belimumab (Benlysta@), besilesomab (Scintimun@), bevacizumab (Avastin@), canakinumab (Ilaris@), catumaxomab (Removab@), cetuximab (Erbitux@), denosumab (Prolia@, Xgeva@), eculizumab (Soliris@), ipilimumab (also known as MDX-010 or MDX-101, Yervoy@), natalizumab (Tysabri@), ofatumumab (Arzerra@), palivizumab 22 WO 2013/186632 PCT/IB2013/001885 (Synagis@), panitumumab (Vectibix@), ranibizumab (Lucentis@), rituximab (Rituxan@, MabThera@), tocilizumab (Actemra@, RoActemra@), trastuzumab (Herceptin@), or ustekinumab (Stelara@) conjugated to functionalized PEG polymers. In some embodiments, a biologically active molecule can be agalsidase alfa (Replagal@ ), agalsidase beta (Fabrazyme@), alglucosidase alfa (Myozyme@), anakinra (Kineret@), bortezomib (Velcade@), conestat alfa (Ruconest@), choriogonadotropin alfa (e.g., Ovidrel@), cinacalcet (Sensipar@), Corifollitropin alfa (Elonva@), dibotermin alfa (InductOs@), eltrombopag (Promacta@), eptotermin alfa (Osigraft@), erlotinib (Tarceva@), follitropin alfa /lutropin alfa, follitropin beta, galsulfase (Naglazyme@), Gefitinib (Iressa@), Human normal immunoglobulin, human normal immunoglobulin (ivig), human normal immunoglobulin (SCIg), idursulfase (Elaprase@), Imiglucerase (Cerezyme@), interferon alfa-2b, interferon beta-i a, interferon beta-lb, Lapatinib (Tykerb@, Tyverb@), Laronidase (Aldurazyme@), Pazopanib (Votrient), pegaptanib, pegfilgrastim, peginterferon alfa-2b, Rasburicase (Elitek@), Reteplase (Retavase@), Somatropin, Sorafenib (Nexavar@), Tenecteplase (TNKase), thyrotropin alfa, Vandetanib (Caprelsa@), or Vemurafenib (Zelboraf@). The conjugates described herein can have an altered pharmacokinetic and pharmacodynamic profile, including, for example, one or more of reduced dosage frequency, extended circulation time, and reduced antigenic properties. As described herein, a biologically active molecule or a derivative thereof can beis conjugated to a functionalized polymer that includes one or more linking groups selected from a phosphotriester, a phosphoramidate, a thiophosphotriester, and a thiophosphoramidate. Suitable functionalized polymers can include different linking groups at each of its termini. A suitable functionalized polymer also can be modified at at least one terminus with a blocking group (e.g., methoxy group) and functionalized at at least another terminus with a linking group as described herein. For example, this document provides a biologically active molecule or a derivative thereof conjugated to functionalized PEG polymers. In some cases, a functionalized PEG polymer has one terminus containing a functional group covalently bound via a phosphotriester, a phosphoramidate, a thiophosphotriester, and a thiophosphoramidate linking group. In some cases, a PEG polymer is a linear PEG polymer (i.e., having two termini). A linear PEG polymer can be functionalized as described herein at one or both termini with the 23 WO 2013/186632 PCT/IB2013/001885 same or different functional group linked via the same or different linking groups. In some cases, one of the termini of a linear PEG polymer is blocked with a blocking group (e.g., methoxy or a protecting group) and the other termini is functionalized with a functional group linked as described herein. In some cases, one of the termini of a linear PEG polymer is functionalized with a phosphotriester linking group and the other termini is functionalized with a phosphoramidate linking group. In other cases, both termini are functionalized with the same or different phosphotriester linking groups. In yet other cases, both termini are functionalized with the same or different phosphoramidate linking groups. Definitions For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. As used herein, the term "about" is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about", whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "alkyl" includes straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.) and branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.). In certain embodiments, a straight chain or branched chain alkyl has twelve or fewer carbon atoms in its backbone (e.g., Ci-12 for straight chain; C3-12 for branched chain). The term Ci-12 includes alkyl groups containing 1 to 12 carbon atoms. The term "alkenyl" includes aliphatic groups that may or may not be substituted, as described above for alkyls, containing at least one double bond and at least two carbon atoms. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, and decenyl) and branched-chain alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has twelve or fewer carbon atoms in its backbone (e.g., C 2
-
1 2 for straight chain; C 3
-
1 2 for branched chain). The term C 2
-
12 includes alkenyl groups containing 2 to 12 carbon atoms. The term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond and 24 WO 2013/186632 PCT/IB2013/001885 two carbon atoms. For example, the term "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl) and branched-chain alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has twelve or fewer carbon atoms in its backbone (e.g., C 2
-
12 for straight chain; C 3 _ 12 for branched chain). The term C 2
-
6 includes alkynyl groups containing 2 to 12 carbon atoms. The term "alkylene" by itself or as part of another molecule means a divalent radical derived from a linear or branched alkane, as exemplified by (-CH 2 -)n, wherein n may be 1 to 24 (e.g., ito 20, Ito 18, Ito 16, Ito 15, Ito 12, Ito 10, ito 8, ito 6, ito 5, ito 4, ito 3, Ito 2, 2 to 24, 2 to 12, 2 to 8). By way of example only, such groups include, but are not limited to, groups having 10 or fewer carbon atoms such as the structures -CH 2
CH
2 - and CH 2
CH
2
CH
2
CH
2 -. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term "alkoxy" is used in its conventional sense, and refers to alkyl groups linked to molecules via an oxygen atom. In some embodiments, an alkoxy has twelve or fewer carbon atoms in its backbone (e.g., a CI- 12 alkoxy). For example, Ci-io, Ci- 8 , CI- 6 , CI-, Ci_ 3 , or CI-2. Non-limiting examples of an alkoxy group include methoxy, ethoxy, propoxy, butoxy, and hexoxy. The term "alkyleneoxyalkylene," as used herein, refers to a divalent radical derived from a linear or branched alkyloxyalkane, as exemplified, but not limited by, -CH 2
-CH
2 -0
CH
2
-CH
2 - and -CH 2
-CH
2 -0-. By way of example only, such groups include, but are not limited to, groups having the formula -(CH 2 )n-O-(CH 2 )m-, wherein n is an integer from 1 to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18) and m is an integer from 0 to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10,1 to6, 1 to2, 1 to3, 1 to4,2 to50,5 to50, 10to50, 15 to50,25 to50,5 to 15,2to 12, 20 to 30, and 6 to 18). The term "oligomeric alkyleneoxyalkylene" refers to p-repetitive alkyleneoxyalkylene wherein p is an integer of between 2 and 24 (e.g., 2 to 20, 2 to 18, 2 to 16, 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3). By way of example only, such groups include, but are not limited to, groups having the formula -((CH 2 )n-O-(CH 2 )m)p-, wherein n is an integer from 1 25 WO 2013/186632 PCT/IB2013/001885 to 50 (e.g., 1 to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., 1 to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 6, 1 to 2, 1 to 3, 1 to 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently an integer from I to 10 (e.g., I to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4 to 10, 6 to 10, 2 to 8, and 3 to 6). In general, the term "arylene" by itself or as part of another molecule means a divalent radical derived from an aryl, including, for example, 5- and 6-membered single-ring aromatic groups, such as benzene and phenyl. Furthermore, the term "arylene" includes a divalent radical derived from a multicyclic aryl group, e.g., tricyclic, bicyclic, such as naphthalene and anthracene. The term "substituted" means that an atom or group of atoms replaces hydrogen as a "substituent" attached to another group. For aryl and heteroaryl groups, the term "substituted", unless otherwise indicated, refers to any level of substitution, namely mono, di, tri, tetra, or penta substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In some cases, two sites of substitution may come together to form a 3-10 membered cycloalkyl or heterocycloalkyl ring. Substituents include, but are not limited to, halo, C 1 -Cio alkyl, C 2 -Cio alkenyl, C 2 -Cio alkynyl, C 1 -Cio alkoxy, C 5
-C
12 aralkyl, C 3
-C
12 cycloalkyl, C 4
-C
12 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C 2
-C
12 alkoxyalkyl, C 5
-C
12 alkoxyaryl, C 5
-C
12 aryloxyalkyl, C 7
-C
12 oxyaryl, C1-C 6 alkylsulfinyl, C 1 -Cio alkylsulfonyl, -(CH 2 )m-0-(C1-Cio alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO 2 , -CN, -NR 9
C(O)
(C
1 -Cio alkyl), -C(O)-(C 1 -Cio alkyl), C 2 -Cio alkthioalkyl, -C(O)O-(C 1 -Cio alkyl), -OH, SO 2 , =S, -COOH, -NR 9 2 , carbonyl, -C(O)-(C 1 -Cio alkyl)-CF 3 , -C(O)-CF 3 , C(O)NR 9 2 , -(C 1 -Cio aryl)-S-(C 6 -Cio aryl), -C(O)-(C 6 -Cio aryl), -(CH 2 )m-0- (CH 2 )m
O-(C
1 -Cio alkyl) wherein each m is from 1 to 8, -C(O)NR 9 2 , -C(S)NR 9 2 , -SO 2
NR
9 2 , NR 9
C(O)NR
9 2 , -NR 9
C(S)NR
9 2 , salts thereof, and the like. Each R9 group in the preceding list independently includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkylaryl. Where substituent groups are specified by their conventional chemical formulas, 26 WO 2013/186632 PCT/IB2013/001885 written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, for example, -CH 2 0- is equivalent to -OCH 2 -. The term "polymer backbone" refers to the main chain of a linear or branched polymer. The term "water-soluble polymer backbone" refers to a polymer backbone having water solubility or dispersibility in water at ambient temperatures and a pH of about 7. For instance, a polyethylene glycol backbone is considered to be water-soluble if the corresponding polyethylene glycol can be solubilized or dispersed in water at ambient temperatures and a pH of about 7. The term "nucleotidic polymer" refers to a single- or double-stranded polymer chain composed of two or more nucleic acids. The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogues of natural nucleotides which have similar binding properties as a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs including, but are not limited to, PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidites, and the like); (iii) conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences. The term "peptidic polymer" refers to a polymer of two or more amino acid residues. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-natural amino acid. As used herein, a "biologically active molecule" includes any molecule which can have a biological effect. Examples of biologically active molecules include therapeutic agents, small molecules, oligo- and polypeptides, oligonucleotides, coding DNA sequences, antisense DNA sequences, mRNAs, antisense RNA sequences, RNAis, and siRNAs, carbohydrates, lipids, growth factors, enzymes, transcription factors, toxins, antigenic peptides (as for vaccines), antibodies, and antibody fragments. The term "group reactive with a biologically active molecule" refers to a functional group that can be covalently bound to a functional group of a biologically active molecule. 27 WO 2013/186632 PCT/IB2013/001885 The terms "protecting group" and "protective group" refer to a moiety that reversibly chemically modifies a functional group in order to obtain chemoselectivity or in order to reduce degradation in one or more subsequent chemical reactions. Suitable protecting groups are well known in the art (see, e.g., Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety). The term "detectable functional group" refers a functional group that physically or chemically interacts with its environment to produce a signal or product that can be detected by analytical and/or imaging methods such as visible, UV-, IR-, NIR-light, X-Ray, and NMR-based imaging methods, enzymatic assays, and UV-, IR-, NMR-, X-ray-, and mass spectrometry-based analytics. As used herein, a "fluorophore" is a chemical group that can be excited by light to emit fluorescence. Some suitable fluorophores may be excited by light to emit phosphorescence. As used herein, a "dye" may include a fluorophore. Non-limiting examples of a fluorophore include: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5 Carboxyfluorescein (5-FAM); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5 Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMIA (5-Carboxytetranethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4 methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino 6-chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Alexa Fluor 350TM; Alexa Fluor 430TM; Alexa Fluor 488TM; Alexa Fluor 532TM; Alexa Fluor 546TM; Alexa Fluor 568TM; Alexa Fluor 594TM; Alexa Fluor 633TM; Alexa Fluor 647TM; Alexa Fluor 660TM; Alexa Fluor 680TM; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAGTM CBQCA; ATTO-TAG T M FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP 28 WO 2013/186632 PCT/IB2013/001885 (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); Blancophor FFG; Blancophor SV; BOBO
TM
-1; BOBO
TM
-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy FL; Bodipy FL ATP; Bodipy FI-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO
TM
-1; BO-PRO
TM
-3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium CrimsonTM; Calcium Green; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue T M ; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP Cyan Fluorescent Protein; CFP/YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine 0; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2
TM
; Cy3.1 8; Cy3.5
TM
; Cy3 T M ; Cy5.1 8; Cy5.5
TM
; Cy5 T M ; Cy7 T M ; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); DiI (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635 NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer -1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43TM; FM 4-46; Fura Red
TM
; Fura Red T M /Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 1OGF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, WV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1; 29 WO 2013/186632 PCT/IB2013/001885 Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf, JC-1; JO-JO-1; JO-PRO-i; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green; Oregon Green 488-X; Oregon GreenTM; Oregon GreenTM 488; Oregon GreenTM 500; Oregon GreenTM 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO- 1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); RsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP
TM
; sgBFP TM (super glow BFP); sgGFP
TM
; sgGFP T M (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-i; SNAFL-2; SNARF calcein; SNARFI; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3 sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 30 WO 2013/186632 PCT/IB2013/001885 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas RedTM; Texas Red-X T M conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. As used herein, a "fluorophore" may include a salt of the fluorophore. Fluorophores may include derivatives that have been modified to facilitate conjugation to another reactive molecule. As such, fluorophores may include amine-reactive derivatives such as isothiocyanate derivatives and/or succinimidyl ester derivatives of the fluorophore. The term "hydrophobic separation handle" refers to a moiety that when attached to or part of a compound reduces the hydrophilicity of that compound, i.e. reduces its tendency to be solved or dispersed in water. The term "separation handle" refers to a moiety that when attached to or part of a compound alters the mobility of that compound in a chromatographic method such that its separation from contaminants is improved. Alternatively, the term "separation handle" refers to a moiety that when attached to or part of a compound improves the yield of the compound in a chromatographic method in comparison to a hydrogen substituent. The term "hydrophobicity" refers to the relative degree with which a compound or moiety is solved or dispersed in a non-aqueous solvent such as n-octanol. The degree of hydrophobicity or hydrophilicity of a compound or moiety can be measured using methods known in the art, such as, reversed phase chromatography and other chromatographic methods, partitioning, accessible surface area methods, and measurement of physical properties such as partial molar heat capacity, transition temperature and surface tension. The term "activating group" refers to a moiety that increases the capability of the group reactive with a biologically active molecule to form a covalent bond with a biologically active molecule. Usually these groups increase or decrease the electronegativity of a selected moiety so 31 WO 2013/186632 PCT/IB2013/001885 it becomes more nucleophilic or more electrophilic. Non-limiting examples of an activating group include: lower alkylamino, diloweralkylamino, amino, halo, aryl, lower alkoxy, lower aralkoxy, aryloxy, mercapto, lower alkylthio, nitro, monophaloalkyl, dihaloalkyl, trihaloalkyl (e.g., CF 3 ), halo, formyl, lower alkanoyl, lower alkylsulfonyl, lower alkylsulfinyl, and the like. The term "essentially pure" refers to chemical purity of a compound provided herein that may be substantially or essentially free of other components which normally accompany or interact with the compound prior to purification. By way of example only, a compound may be "essentially pure" when the preparation of the compound contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, an "essentially pure" compound may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. For the purposes of this document, preparations of functionalized polymers or conjugates differing only in the length of their polymer chain are considered to be essentially pure. By way of example a preparation of a mono-functionalized compound may be "essentially pure" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating unfunctionalized and/or poly-functionalized polymers. An essentially pure compound may be obtained using chromatographic purification methods. The term "protic solvent" is used herein to refer to solvents which comprise dissociable hydrogen ions. Examples of protic solvents include alcohols, such as ethanol, and methanol. The phrase "pharmaceutically acceptable" is used herein to refer to those inhibitors, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Some of the compounds provided herein are acidic and may form a salt with a pharmaceutically acceptable cation. Some of the compounds herein can be basic and accordingly, may form a salt with a pharmaceutically acceptable anion. All such salts, including 32 WO 2013/186632 PCT/IB2013/001885 di-salts are within the scope of the compositions described herein and they can be prepared by conventional methods. For example, salts can be prepared by contacting the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization. Salts, for example, include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4 hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; and (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. Compounds The following compounds are suitable for conjugating to a biologically active molecule or a derivative thereof. Compounds offormula (1) 33 WO 2013/186632 PCT/IB2013/001885 Provided herein is a compound comprising a water-soluble, non-nucleotidic and non peptidic polymer backbone having at least one terminus covalently bonded to a structure of formula (1): E R1
A-O-P-Z
1 -L-M-R z2 K I G linking group or pharmaceutical salt thereof wherein: A is the point of bonding to a terminus of the polymer backbone, E is an oxygen or sulfur atom, K is selected from the group consisting of alkylene, alkyleneoxyalkylene, or an oligomeric form of alkyleneoxyalkylene, G is selected from the group consisting of hydrogen, an alkoxy, or a hydrophobic separation handle, Z' and Z 2 can be oxygen or nitrogen, in such way that both Z' and Z 2 may be oxygen, but when Z' is NH then Z 2is oxygen, and when Z 2 is NH then Z' is oxygen, L is selected from the group consisting of a divalent radical of a nucleoside, linear alkylene, branched alkylene, alkyleneoxyalkylene, oligomeric form of alkyleneoxyalkylene, arylene, and substituted arylene, M is a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof, a group reactive with a biologically active molecule or a derivative thereof, or detectable functional group, R is a protecting group, activating group, hydrogen or absent. A polymer backbone, as provided herein, can be branched or linear. For example, a polymer backbone can have from 2 to 100 termini (e.g., 2 to 80, 2 to 75, 2 to 60, 2 to 50, 2 to 40, 2 to 35, 2 to 25, 2 to 10, 2 to 5, 4 to 20, 5 to 25, 10 to 50, 25 to 75, 3 to 6, 5 to 15 termini). In some embodiments, a polymer is linear and therefore has 2 termini. In some embodiments, only one termini of a polymer backbone is covalently bonded to the structure of formula (1). In some embodiments, wherein a polymer has two termini, both termini of the polymer backbone are covalently bonded to the structure of formula (1). 34 WO 2013/186632 PCT/IB2013/001885 A polymer backbone can be, for example, poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol), polyoxazoline, or a copolymer thereof. A polyalkylene glycol includes linear or branched polymeric polyether polyols. Such polyalkylene glycols, include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have average molecular weights between about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, between about 500 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 500 Da and about 100,000 Da. For example, a polymer used herein can have a molecular weight of about 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, and 500 Da. In some embodiments, the molecular weight of the polymer is between about 500 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 500 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, a polymer backbone is a linear or branched poly(ethylene glycol). In some embodiments, the poly(ethylene glycol) molecule is a linear polymer. The molecular weight of the linear chain PEG may be between about 1,000 Da and about 100,000 Da. For example, a linear chain PEG used herein can have a molecular weight of about 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the linear chain PEG is 35 WO 2013/186632 PCT/IB2013/001885 between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the linear chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the linear chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the linear chain PEG is between about 5,000 Da and about 20,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG may be between about 1,000 Da and about 100,000 Da. For example, a branched chain PEG used herein can have a molecular weight of about 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da. In some embodiments, E is oxygen. In some embodiments, E is sulfur. In some embodiments, K is a linear or branched alkylene. For example, K can be selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene. In some embodiments, K can be an alkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, K can be a residue from diethylene glycol, triethylene glycol, tetraethylene glycol, or hexaethylene glycol. In some embodiments, K is selected from the group consisting of -(CH 2 )n- and -((CH 2 )n-O-(CH 2 )m)p-, wherein n is an integer from I to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, Ito 10, ito 6, ito 6, ito 2, ito 3, ito 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, Ito 18, Ito 15, Ito 12, Ito 10, 1 to6, I to2, I to3, I to4,2to5O,5to5O, 10to50, 15to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently an integer from Ito 10(e.g., 1 to8, 1 to6, Ito5, Ito3,2to 10,4to 10,6to 10,2to8,and3to6). 36 WO 2013/186632 PCT/IB2013/001885 In some embodiments, G is a hydrophobic separation handle. For example, G can be a substituted or unsubstituted trityloxy group. In some embodiments, G is selected from the group consisting of monoalkoxy substituted trityloxy group or dialkoxy substituted trityloxy group. In some embodiments, one of Z' and Z 2 is NH and the other is 0. For example, Z' is 0 and Z2 is NH; Z is NH and Z 2 is 0. In some embodiments, both Z' and Z2 are 0. In some embodiments, L is a linear or branched alkylene. For example, L can be selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene. In some embodiments, L can be an alkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, L can be a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, L is selected from the group consisting of -(CH 2 )n- and -((CH 2 )n-O-(CH 2 )m)p-, wherein n is an integer from I to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, Ito 10, ito 6, ito 6, ito 2, ito 3, ito 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20,Ito 18,Ito 15,Ito 12,Ito l0,1to6,to2,to3,to4,2to5,5to5,10to50,i15to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently an integer from Ito 10(e.g., I to8, I to6, ito5, ito3,2to 10,4to 10,6to 10,2to8,and3to6). In some embodiments, L is a substituted or unsubstituted arylene. For example, L can be a structure with the formula: Wr where W is a substituent and r is an integer from 0 to 4. For example, W can be selected from the group consisting of: halo, C1-Cio alkyl, C 2 -Cio alkenyl, C 2 -Cio alkynyl, C 1 -Cio alkoxy,
C
5
-C
12 aralkyl, C 3
-C
12 cycloalkyl, C 4
-C
12 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C 2
-C
12 alkoxyalkyl, C 5
-C
1 2 alkoxyaryl, C 5
-C
1 2 aryloxyalkyl, C 7
-C
1 2 oxyaryl,
C
1
-C
6 alkylsulfinyl, C 1 -Cio alkylsulfonyl, -(CH 2 )m-0-(C 1 -Cio alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO 2 , -CN, -NR 9
C(O)-(C
1 -Cio alkyl), -C(O)-(C 1 -Cio 37 WO 2013/186632 PCT/IB2013/001885 alkyl), C 2 -Cio alkthioalkyl, -C(O)O-(C 1 -Cio alkyl), -OH, -SO 2 , =S, -COOH, -NR9 2 , carbonyl, -C(O)-(C 1 -Cio alkyl)-CF 3 , -C(O)-CF 3 , -C(O)NR 9 2 , -(C 1 -Cio aryl)-S-(C 6 -Cio aryl), -C(O)-(C 6 -Cio aryl), -(CH 2 )m-0-(CH 2 )m-0- (C 1
-C
1 O alkyl) wherein each m is from 1 to 8, -C(O)NR 9 2 , -C(S)NR 9 2 , -S0 2
NR
9 2 , -NR 9
C(O)NR
9 2 , -NR 9
C(S)NR
9 2 , salts thereof, and the like. Each R 9 group in the preceding list independently includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkylaryl. In some embodiments, W is R 1 as described above. Non-limiting examples of L include: w w wr Wr r r I r Wr In some cases, L can also be a divalent radical of a nucleoside. For example, L can be a divalent radical of a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, 5-methyluridine, thymidine, uridine, deoxyuridine, cytidine, and deoxycytidine. M is a group reactive with a biologically active molecule or a derivative thereof and can be selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, and iodoacetamide. In some embodiments, the group is protected or further reacted with a group R as shown in the structure of formula (1). The point of attachment of such a group is well understood by those of skill in the art. In some embodiments, R is absent. In some embodiments, R is a protecting group. For this purpose, R may include any suitable protecting group based on the group to be protected. 38 WO 2013/186632 PCT/IB2013/001885 For example, R may include any suitable hydroxyl functional group including, but not limited to, ether, ester, carbonate, or sulfonate protecting groups. In particular, the ether protecting group may include benzyloxymethyl (BOM), methylthiomethyl (MTM), phenylthiomethyl (PTM), , cyanoethyl, 2,2-dichloro- 1,1 difluoroethyl, 2-chloroethyl, 2-bromoethyl, tetrahydropyranyl (THP), , phenacyl, 4 bromophenacyl, , allyl, propargyl, , t-butyl, benzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl (MPM-OAr), o-nitrobenzyl, 2,6-dichlorobenzyl, 3,4-dichlorobenzyl, 4 (dimethylamino)carbonylbenzyl, 4-methylsulfinylbenzyl (Msib), 9-anthrylemethyl, 4-picolyl, heptafluoro-p-tolyl, tetrafluoro-4-pyridyl, trimethylsilyl (TMS), , and protecting groups. The ester protecting group may include acetoxy (OAc), aryl formate, acetate, levulinate, pivaloate, benzoate, and 9-fluoroenecarboxylate. In one embodiment, the ester protecting group is an acetoxy group. The carbonate protecting group may include aryl methyl carbonate, 1 -adamantyl carbonate (Adoc-OAr), t-butyl carbonate (BOC-OAr), 4-methylsulfinylbenzyl carbonate (Msz OAr), 2,4-dimethylpent-3-yl carbonate (Doc-OAr), aryl 2,2,2-trichloroethyl carbonate,. The sulfonate protecting groups may include aryl methanesulfonate, aryl toluenesulfonate, and aryl 2-formylbenzenesulfonate. In some embodiments, R may include any suitable amino protecting group, including, but not limited to, carbamate, amide, N-alkyl, or N-aryl-derived protecting groups. In particular, the carbamate protecting group may include, for example, 9 fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), carboxybenzyl carbamate (cbz), methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7 dibromo)fluorenylmethyl carbamate, 17-tetrabenzol[a,c,g,i]fluorenylmethyl carbamate (Tbfmoc), 2-chloro-3-indenylmethyl carbamate (Climoc), 2,7-di-t-butyl[9-(10,10-dioxo 10,10,10,1 0-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 1,1 dioxobenzo[b]thiophene-2-ylmethyl carbamate (Bsmoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2-haloethyl carbamate, 1,1 -dimethyl-2,2-dibromoethyl carbamate (DB-t-boc), 1,1 -dimethyl-2,2,2 trichloroethyl carbamate (TCBoc), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di t-butylphenyl)- 1 -methylethyl carbamate (t-Bumeoc), N-2-pivaloylamino)- 1,1 -dimethylethyl 39 WO 2013/186632 PCT/IB2013/001885 carbamate, 2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate (NpSSPeoc), 2-(N,N dicycloheylcarboxamido)ethyl carbamate, 1-adamanyl carbamate (1-Adoc), cinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc), 4-nicrocinnamyl carbamate (Noc), 3-(3'pyridyl)prop 2-enyl carbamate (Paloc), 8-quinolyl carbamate, alkyldithio carbamate, p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate (Pnz), p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(P toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4 methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2 phosphonioethyl carbamate (Peoc), 1,1-dimethyl-2-cyanoethyl carbamate, 2-(4-nitrophenyl)ethyl carbamate, 4-phenylacetoxybenzyl carbamate (PhAcOZ), and m-chloro-p-acyloxybenzyl carbamate. In some embodiments, the carbamate protecting group is chosen from 9 fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz). The amide protecting group may include, for example, acetamide, phenylacetamide, 3 phenylpropanamide, pent-4-enamide, picolinamide, 3-pyridylcarboxamide, benzamide, p phenylbenzamide, 2-methyl-2-(o-phenylazophenoxy)propanamide), 4-chlorobutanamide, acetoacetamide, 3-(p-hydroxyphenyl)propanamide), and (N' dithiobenzyloxycarbonylamino)acetamide. Examples of suitable protecting groups also include tert-butyl, benzyl, 4-methoxybenzyl, benzyloxymethyl, phenacyl, allyl, trimethylsilyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acetal and ketal derivatives. In some embodiments, R is selected from trityls, substituted trityls (e.g., monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), trimethoxytrityl (TMTr), 2 chlorotrityl (ClTr) and p-bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, for example, U.S. Publication No. 2007/0276139). In some embodiments, R is selected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal. In some embodiments, R 1 is a hydrophobic separation handle. A hydrophobic separation handle is as described herein. In some embodiments, the hydrophobic separation handle is also a protecting group as described herein. In some 40 WO 2013/186632 PCT/IB2013/001885 embodiments, at least one of R, R 1 , and G is a hydrophobic separation handle. For example, only one of R, R 1 , and G is a hydrophobic separation handle. In some embodiments, only one of R 1 , R and G is a hydrophobic separation handle (e.g., a trityl group) as provided herein. For example, if R is a hydrophobic separation handle, then R 1 is absent and G is hydrogen or an alkoxy. In some embodiments, R is absent or a protecting group, R 1 is a hydrophobic separation handle, and G is hydrogen or an alkoxy. In some embodiments, wherein more than one of R, R 1 and G is a hydrophobic separation handle, one of R, R I and G is more hydrophobic than the others (e.g., substantially more hydrophobic). In some embodiments, the hydrophobic separation handle is a substituted or unsubstituted trityl or trityloxy group. For example, only one of R, R 1 and G is a substituted or unsubstituted trityl or trityloxy group. A compound as described above can be prepared, for example, by contacting a water soluble, non-peptidic and non-nucleotidic polymer, in a water-free solvent (e.g., an organic solvent), with a reagent of formula (4): R1 N-P-ZL-M-R R 6 Z2 K-G wherein:
R
5 and R 6 independently from each other represent C 1
-C
6 -alkyl or R 5 and R6 jointly form a 5- or 6-membered ring with the N to which they are bonded. In some embodiments, R 5 and R6 are independently a C1-C 6 -alkyl. For example, R 5 and R 6 can be independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and hexyl. In some embodiments, R 5 and R 6 are isopropyl. In some embodiments, R 5 and R6 can jointly form a 5- or 6-membered ring with the N to which they are bonded. For example, R 5 and R6 jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl, particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, 41 WO 2013/186632 PCT/IB2013/001885 particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, or pyrazolyl, particularly 3- and 5-pyrazolyl. In some embodiments, R 5 and R6 can jointly form a morpholine ring. The ratio of a polymer to a reagent of formula (4) can range from about 1:10 to about 10:1 (e.g., about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:81 about 1:9, about 2:8, about 3:7, about 4:6 about 5:10, and about 4:8). In some embodiments, the ratio of a polymer to a reagent of formula (4) is from about 1:1 to about 1:10, for example, about 2:1. An activating reagent can then be added to the mixture of the polymer and the reagent of formula (4). An activating reagent can be any group suitable to initiate coupling of the polymer and the reagent of formula (4). Suitable activating reagents include, for example, 1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT), 5-(benzylthio)-1H-tetrazole (BTT), Activator 42 (5-(3,5 bis(trifluoromethyl)phenyl)-1H-tetrazole), 2-ethylthiotetrazole, 2-bezylthiotetrazole, 4,5 dicyanoimidazole and 4,5-dicyanoimidazole (DCI). In some embodiments, an activating agent can be selected from pyridinium hydrochloride, pyridinium trifluoroacetage, and buffered carboxylic acids. An oxidizing agent can then be added to oxidize p 3 to p 5 . Suitable oxidizing agents and conditions can be readily determined by those of ordinary skill in the art. For example, an oxidant such as RuO 4 /NMO, Dess-Martin's reagent, DMSO/triflic anhydride, PDC, hydrogen peroxide, inorganic peroxides, nitric acid, nitrates, chlorite, chlorate, perchlorate, hypochlorite, peroxides, iodine, ozone, nitrous oxide, silver oxide, permanganate salts, hexavalent chromium compounds, chromic acid, dichromic acids, chromium trioxide, pyridinium chlorochromate, persulfuric acid, sulfoxides, sulfuric acid, Tollens' reagent, 2,2'-dipyridiyldisulfide (DPS), and osmium tetroxide may be used. In some embodiments, iodine can be used as an oxidizing agent. For example, a solution of iodine can be used and prepared by dissolving iodine in a mixture of pyridine, tetrahydrofuran and water. Elemental sulfur can be used for phosphite oxidation combined with formation of sulfurized product. In some embodiments, other more soluble and more reactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide (Beaucage reagent), phenylacetyl disulfide (PADS), or dimethylthiuram (DTD) can be used. Alternatively, peroxides exemplified by t-butyl hydrogen peroxide or m-chlorobenzoyl peroxide may be used for p 3 to p 5 oxidation. 42 WO 2013/186632 PCT/IB2013/001885 In some embodiments, an oxidizing reagent is selected from a group consisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide, acetone peroxide, sulfur, and thiuram disulfide. In some embodiments, R is a protecting group or a hydrophobic separation handle and the method can include purifying the compound using chromatography (e.g., reverse phase chromatography). In some embodiments, the method also includes removing the protecting group. For the methods provided above, the deprotection may involve, for example, either sequential or one-pot deprotection of certain protecting groups. Suitable reagents and conditions for the deprotection can be readily determined by those of ordinary skill in the art. For example, deprotection may be achieved upon treatment of the protected compound under conditions so that hydroxyl protecting groups, such as acetate, isopropylidine, benzylidine, trityl, and/or pixyl protecting groups, are removed from the protected compound. The acetate group can be cleaved under mild conditions, for example, by diluted solution of ammonia or by solution of potassium carbonate. The benzylidene and isopropylidene groups can be cleaved by hydrogenation or using acidic hydrolysis as described elsewhere by R.M. Hann et al., J. Am. Chem. Soc., 72, 561 (1950). In yet another example, the deprotection can be performed so that amino protecting groups, such as 9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz) protecting groups are cleaved from the protected compound. 9 fluorenylmethyl carbamate (FMOC) can be removed under mild conditions with an amine base (e.g., piperidine) to afford the free amine and dibenzofulvene, as described by E. Atherton et al., "The Fluorenylmethoxycarbonyl Amino Protecting Group," in The Peptides, S. Udenfriend and J. Meienhofer, Academic Press, New York, 1987, p. 1. t-butyl carbamate (Boc) can be removed, as reported by G.L. Stahl et al., J. Org. Chem., 43, 2285 (1978), under acidic conditions (e.g., 3 M HCl in EtOAc). Hydrogenation can be used to cleave the carboxybenzyl carbamate (cbz) protecting group as described by J. Meienhofer et al., Tetrahedron Lett., 29, 2983 (1988). In some embodiments, deprotection may be performed under anaerobic conditions. The deprotection may also be performed at ambient temperature or at temperatures of from about 20 - 60 0 C (e.g., 25, 30, 35, 40, 45, 50, or 55 0 C). 43 WO 2013/186632 PCT/IB2013/001885 In some cases, a compound as described above can be further purified using precipitation and/or crystallization. Compounds offormula (2) Also provided herein are compounds of formula (2): El E
R
1
-M
1
-L
1
-Z
3 -- P-- 0 polymer O--P- Z 1 -L-M- R I G Z 4 '-- z2 Gi--K1 K-G linking group linking group or a salt form thereof, wherein: polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer backbone, wherein each linking group is bonded at a different terminus of said polymer; E and El are independently 0 or S; K and K 1 are independently selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G and G 1 are independently absent or are selected from the group consisting of: alkoxy and a hydrophobic separation handle; each pair of Z 1 and Z 2 and Z 3 and Z 4 are independently selected from 0 and NH, wherein only one of each pair of Z' and Z 2 and Z 3 and Z 4 can be NH; L and L' are independently selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; M and M 1 are independently selected from a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof, a group reactive with a biologically active molecule or a derivative thereof, or is a detectable functional group, wherein 44 WO 2013/186632 PCT/IB2013/001885 M and M 1 are different, and wherein at least one of M and M1 is a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof or a group reactive with a biologically active molecule or a derivative thereof ; and R and R 1 are independently absent, hydrogen, a protecting group, or an activating group; wherein when M is a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof, then R is a protecting group or a hydrophobic separation handle; wherein when M is a group reactive with a biologically active molecule or a derivative thereof, R is absent, hydrogen, or an activating group; wherein when M is a detectable functional group, R is absent or hydrogen; wherein when M 1 is a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof, then R 1 is a protecting group or a hydrophobic separation handle; wherein when M 1 is a group reactive with a biologically active molecule or a derivative thereof, R 1 is absent, hydrogen, or an activating group; and wherein when M 1 is a detectable functional group, R 1 is absent or hydrogen. A polymer can be, for example, poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol), polyoxazoline, or a copolymer thereof. Such polyalkylene glycols, include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have average molecular weights between about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, between about 500 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 500 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, and 45 WO 2013/186632 PCT/IB2013/001885 500 Da. In some embodiments, the molecular weight of the polymer is between about 500 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 500 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the polymer is a poly(ethylene glycol) polymer. The molecular weight of the PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the n PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the PEG is between about 5,000 Da and about 20,000 Da. In some embodiments, El is oxygen. In some embodiments, El is sulfur. In some embodiments, E2 is oxygen. In some embodiments, E 2 is sulfur. In some embodiments, both of E and E 2 are oxygen. In some embodiments, K and K' are independently selected from a linear or branched alkylene. For example, K and K' can be independently selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene. In some embodiments, K and K' are independently an alkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, K and K' can be independently a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, K and K' are independently selected from the group consisting of -(CH 2 )n 1 - and ((CH 2 )n-O-(CH 2 )m)p-, wherein n is an integer from I to 50 (e.g., 1 to 40,1 to 30, 1 to 25, 1 to 20, Ito 18, Ito 15, Ito 12, Ito 10, 1to6, 1to6, 1to2, 1to3, 1to4,2to5O,5to5O, 10to50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., I to 40,lto3O, 1to25, 1to2O, 1to 18, 1to 15, 1to 12, 1to 10, 1to6, 1to2, 1to3, 1to4,2to5O, 46 WO 2013/186632 PCT/IB2013/001885 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently an integer from I to 10 (e.g., I to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4 to 10, 6 to 10, 2 to 8, and 3 to 6). In some embodiments, G and G 1 are independently a hydrophobic separation handle. For example, G and G 1 are independently a substituted or unsubstituted trityloxy group. In some embodiments, G and G 1 are independently selected from the group consisting of monoalkoxy substituted trityloxy group or dialkoxy substituted trityloxy group. In some embodiments, one of Z' and Z 2 is NH and the other is 0. For example, Z' is 0 and Z 2 is NH; Z' is NH and Z 2 is 0. In some embodiments, both Z' and Z 2 are 0. In some embodiments, one of Z' and Z 2 is NH and the other is 0. For example, Z 3 is 0 and Z 4 is NH; Z 3 is NH and Z 4 is 0. In some embodiments, both Z 3 and Z 4 are 0. In some embodiments, one of ZI and Z 2 and Z 3 and Z 4 is NH and the other is 0. For example, Z' and Z 3 are 0 and Z 2 and Z 4 are NH; Z' and Z 3 are NH and Z 2 and Z 4 0. In some embodiments, Z' and Z 3 are 0 and Z 2 and
Z
4 are 0. In some embodiments, L and L' are independently selected from a linear or branched alkyl. For example, L and L' can be independently selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene. In some embodiments, L and L' are independently an alkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, L and L' can be independently a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, L and L' are independently selected from the group consisting of -(CH 2 )n 1 - and ((CH 2 )n-O-(CH 2 )m)p-, wherein n is an integer from I to 50 (e.g., 1 to 40,1 to 30, 1 to 25, 1 to 20, Ito 18, Ito 15, Ito 12, Ito 10, Ito6, Ito6, Ito2, Ito3, Ito4,2to5O,5to5O, 10to50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., I to 40,1to3O, Ito25, Ito2O, to 18, Ito 15, Ito 12, Ito 10, Ito6, Ito2, Ito3, Ito4,2to5O, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently an integer from I to 10 (e.g., I to 8, 1 to 6, 1 to 5, 1 to 3, 2 to 10, 4 to 10, 6 to 10, 2 to 8, and 3 to 6). In some embodiments, L and L' are independently a substituted or unsubstituted arylene. For example, L and L' can be independently a structure with the formula: 47 WO 2013/186632 PCT/IB2013/001885 Wr wherein W is a substituent and r is an integer from 0 to 4. For example, W can be selected from the group consisting of: halo, C 1 -Cio alkyl, C 2 -Cio alkenyl, C 2 -Cio alkynyl, C 1 -Cio alkoxy, C 5
-C
12 aralkyl, C 3
-C
12 cycloalkyl, C 4
-C
12 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C 2
-C
12 alkoxyalkyl, C 5
-C
12 alkoxyaryl, C 5
-C
12 aryloxyalkyl, C 7
-C
12 oxyaryl, C1-C 6 alkylsulfinyl, CI-Cio alkylsulfonyl, -(CH 2 )m-0-(C 1 -Cio alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO 2 , -CN, -NR 9
C(O)-(C
1
-C
1 O alkyl), C(O)-(C 1 -Cio alkyl), C 2 -Cio alkthioalkyl, -C(O)O-(C 1 -Cio alkyl), -OH, -SO 2 , =S, COOH, -NR9 2 , carbonyl, -C(O)-(C 1 -Cio alkyl)-CF 3 , -C(O)-CF 3 , -C(O)NR 9 2 , -(C 1 -Cio aryl)-S-(C 6 -Cio aryl), -C(O)-(C 6 -Cio aryl), -(CH 2 )m-0-(CH 2 )m-0-(C 1
-C
1 O alkyl) wherein each m is from 1 to 8, -C(O)NR 9 2 , -C(S)NR 9 2 , -S0 2
NR
9 2 , -NR 9
C(O)NR
9 2 , NR 9
C(S)NR
9 2 , salts thereof, and the like. Each R9 group in the preceding list independently includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkylaryl. In some embodiments, W is R 1 as described above. Non-limiting examples of L and L' include: r r r Wr 48 WO 2013/186632 PCT/IB2013/001885 L and Ll can also independently be a divalent radical of a nucleoside. For example, L and Ll can be a divalent radical of a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, 5-methyluridine, thymidine, uridine, deoxyuridine, cytidine, and deoxycytidine. M is a group reactive with a biologically active molecule or a derivative thereof and can be selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, and iodoacetamide. In some embodiments, the group is protected or further reacted with a group R and R 1 as shown in the formula (2). The point of attachment of such a group is well understood by those of skill in the art. In some embodiments, M or M 1 is a detectable functional group. A detectable functional group, as used herein, can be any chemical or substance which is used to provide a signal or contrast in imaging. The signal enhancing domain can be an organic molecule, metal ion, salt or chelate, particle (particularly iron particle), or labeled peptide, protein, polymer or liposome. For example, a detectable functional group can include one or more of a radionuclide, a paramagnetic metal, a fluorophore, a dye, and an enzyme substrate. In some embodiments, a detectable functional group is biotin or a fluorophore. In some embodiments, the detectable functional group is a physiologically compatible metal chelate compound consisting of one or more cyclic or acyclic organic chelating agents complexed to one or more metal ions with atomic numbers 21-29, 42, 44, or 57-83. For x-ray imaging, the detectable functional group may consist of iodinated organic molecules or chelates of heavy metal ions of atomic numbers 57 to 83. In some embodiments, the detectable functional group is mI-IgG. Examples of suitable compounds are described in M. Sovak, ed., "Radiocontrast Agents," Springer-Verlag, pp.23-125 (1984) and U.S. Pat. No. 4,647,447. For ultrasound imaging, the detectable functional group can consist of gas-filled bubbles such as Albunex, Echovist, or Levovist, or particles or metal chelates where the metal ions have atomic numbers 21-29, 42, 44 or 57-83. Examples of suitable compounds are described in Tyler et al., Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P. Swanson, "Enhancement Agents for Ultrasound: Fundamentals," Pharmaceuticals in Medical Imaging, pp. 682-87. (1990). 49 WO 2013/186632 PCT/IB2013/001885 For nuclear radiopharmaceutical imaging or radiotherapy, the detectable functional group can consist of a radioactive molecule. In some embodiments, the chelates of Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In, and Ga can be used. In some embodiments, the chelates of Tc-99m can be used. Examples of suitable compounds are described in Rayudu GVS, Radiotracers for Medical Applications, I, pp. 201 and D. P. Swanson et al., ed., Pharmaceuticals in Medical Imaging, pp. 279-644 (1990). For ultraviolet/visible/infrared light imaging, the detectable functional group can consist of any organic or inorganic dye or any metal chelate. For MRI, the detectable functional group can consist of a metal-ligand complex of a paramagnetic form of a metal ion with atomic numbers 21-29, 42, 44, or 57-83. In some embodiments, the paramagnetic metal is chosen from: Gd(III), Fe(III), Mn(II and III), Cr(III), Cu(II), Dy(III), Tb(III), Ho(III), Er(III) and Eu(III). Many suitable chelating ligands for MRI agents are known in the art. These can also be used for metal chelates for other forms of biological imaging. For example, an imaging agent can include: Gadovist, Magnevist, Dotarem, Omniscan, and ProHance. In some embodiments, R and/or R 1 is absent. In some embodiments, R and/or R1 is a protecting group. For this purpose, R and/or R 1 may include any suitable protecting group based on the group to be protected. For example, R and/or R 1 may include any suitable hydroxyl functional group including, but not limited to, ether, ester, carbonate, or sulfonate protecting groups. In particular, the ether protecting group may include benzyloxymethyl (BOM), methylthiomethyl (MTM), phenylthiomethyl (PTM), , cyanoethyl, 2,2-dichloro- 1,1 difluoroethyl, 2-chloroethyl, 2-bromoethyl, tetrahydropyranyl (THP), , phenacyl, 4 bromophenacyl, , allyl, propargyl, , t-butyl, benzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl (MPM-OAr), o-nitrobenzyl, 2,6-dichlorobenzyl, 3,4-dichlorobenzyl, 4 (dimethylamino)carbonylbenzyl, 4-methylsulfinylbenzyl (Msib), 9-anthrylemethyl, 4-picolyl, heptafluoro-p-tolyl, tetrafluoro-4-pyridyl, trimethylsilyl (TMS), , and protecting groups. The ester protecting group may include acetoxy (OAc), aryl formate, acetate, levulinate, pivaloate, benzoate, and 9-fluoroenecarboxylate. In one embodiment, the ester protecting group is an acetoxy group. 50 WO 2013/186632 PCT/IB2013/001885 The carbonate protecting group may include aryl methyl carbonate, 1 -adamantyl carbonate (Adoc-OAr), t-butyl carbonate (BOC-OAr), 4-methylsulfinylbenzyl carbonate (Msz OAr), 2,4-dimethylpent-3-yl carbonate (Doc-OAr), aryl 2,2,2-trichloroethyl carbonate,. The sulfonate protecting groups may include aryl methanesulfonate, aryl toluenesulfonate, and aryl 2-formylbenzenesulfonate. In some embodiments, R may include any suitable amino protecting group, including, but not limited to, carbamate, amide, N-alkyl, or N-aryl-derived protecting groups. In particular, the carbamate protecting group may include, for example, 9 fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), carboxybenzyl carbamate (cbz), methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7 dibromo)fluorenylmethyl carbamate, 17-tetrabenzol[a,c,g,i]fluorenylmethyl carbamate (Tbfmoc), 2-chloro-3-indenylmethyl carbamate (Climoc), 2,7-di-t-butyl[9-(10,10-dioxo 10,10,10,1 0-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 1,1 dioxobenzo[b]thiophene-2-ylmethyl carbamate (Bsmoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2-haloethyl carbamate, 1,1 -dimethyl-2,2-dibromoethyl carbamate (DB-t-boc), 1,1 -dimethyl-2,2,2 trichloroethyl carbamate (TCBoc), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di t-butylphenyl)- 1 -methylethyl carbamate (t-Bumeoc), N-2-pivaloylamino)- 1,1 -dimethylethyl carbamate, 2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate (NpSSPeoc), 2-(N,N dicycloheylcarboxamido)ethyl carbamate, 1-adamanyl carbamate (1-Adoc), cinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc), 4-nicrocinnamyl carbamate (Noc), 3-(3'pyridyl)prop 2-enyl carbamate (Paloc), 8-quinolyl carbamate, alkyldithio carbamate, p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate (Pnz), p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(P toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4 methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2 phosphonioethyl carbamate (Peoc), 1,1-dimethyl-2-cyanoethyl carbamate, 2-(4-nitrophenyl)ethyl carbamate, 4-phenylacetoxybenzyl carbamate (PhAcOZ), and m-chloro-p-acyloxybenzyl carbamate. In some embodiments, the carbamate protecting group is chosen from 9 51 WO 2013/186632 PCT/IB2013/001885 fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz). The amide protecting group may include, for example, acetamide, phenylacetamide, 3 phenylpropanamide, pent-4-enamide, picolinamide, 3-pyridylcarboxamide, benzamide, p phenylbenzamide, 2-methyl-2-(o-phenylazophenoxy)propanamide), 4-chlorobutanamide, acetoacetamide, 3-(p-hydroxyphenyl)propanamide), and (N' dithiobenzyloxycarbonylamino)acetamide. Examples of suitable protecting groups also include tert-butyl, benzyl, 4-methoxybenzyl, benzyloxymethyl, phenacyl, allyl, trimethylsilyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acetal and ketal derivatives. In some embodiments, R is selected from trityls, substituted trityls (e.g., monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), trimethoxytrityl (TMTr), 2 chlorotrityl (ClTr) and p-bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, for example, U.S. Publication No. 2007/0276139). In some embodiments, R is selected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal. In some embodiments, R and/or R 1 is a hydrophobic separation handle. A hydrophobic separation handle is as described herein. In some embodiments, the hydrophobic separation handle is also a protecting group as described herein. In some embodiments, at least one of R, R 1 , G, and G 1 is a hydrophobic separation handle. In some embodiments, only one of R and G and R 1 and G 1 is a hydrophobic separation handle (e.g., a trityl group) as provided herein. For example, if R is a hydrophobic separation handle, then G is hydrogen or an alkoxy. Alternatively, if R 1 is a hydrophobic separation handle, then G 1 is absent or an alkoxy. In some embodiments, R is absent or a protecting group, R 1 is a hydrophobic separation handle, and G and G 1 are independently absent or an alkoxy, wherein R 1 is more hydrophobic than R. In some embodiments, R 1 is absent or a protecting group, R is a hydrophobic separation handle, and G and G 1 are independently absent or an alkoxy, wherein R is more hydrophobic than R1. In some embodiments, the hydrophobic separation handle is a substituted or unsubstituted trityl or trityloxy group. For example, only one of R, R 1 , G, and G 1 is a substituted or unsubstituted trityl or trityloxy group. 52 WO 2013/186632 PCT/IB2013/001885 A compound as described above can be prepared, for example, by contacting a water soluble, non-peptidic and non-nucleotidic polymer, in a water-free solvent (e.g., an organic solvent), with a reagent selected from formula (5): Rs5
N-P-Z
1 -L-M-R R6/ 1 Z2 K-G wherein:
R
5 and R 6 independently from each other represent C 1
-C
6 -alkyl or R 5 and Ri jointly form a 5- or 6-membered ring with the N to which they are bonded, and formula (6): R 7
N-P-Z
3
-L
1
-M
1 -R1
R
8 1 Z4
K
1 -G1 wherein: R and R 8 independently from each other represent C 1
-C
6 -alkylor R7 and R 8 jointly form a 5- or 6-membered ring with the N to which they are bonded; under conditions that facilitate formation of monoderivatized product. In some embodiments, R 5 and R6 are independently a C1-C 6 -alkyl. For example, R 5 and R6 can be independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and hexyl. In some embodiments, R 5 and R 6 are isopropyl. In some embodiments, R 5 and R6 jointly form a 5- or 6-membered ring with the N to which they are bonded. For example, R 5 and R6 jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl, particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, and pyrazolyl, particularly 3- and 5-pyrazolyl. In some embodiments, R 5 and R6 jointly form a morpholine ring. In some embodiments, R 7 and R 8 are independently a C 1
-C
6 -alkyl. For example, R 7 and R can be independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, 53 WO 2013/186632 PCT/IB2013/001885 butyl, isobutyl, sec-butyl, tert-butyl, and hexyl. In some embodiments, R 7 and R 8 are isopropyl. In some embodiments, R 7 and R 8 jointly form a 5- or 6-membered ring with the N to which they are bonded. For example, R7 and R 8 jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl, particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, and pyrazolyl, particularly 3- and 5-pyrazolyl. In some embodiments, R 7 and R 8 jointly form a morpholine ring. The ratio of a polymer to a reagent of formula (5) or (6) can range from about 1:10 to about 10:1 (e.g., about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:81 about 1:9, about 2:8, about 3:7, about 4:6 about 5:10, and about 4:8). In some embodiments, the ratio of a polymer to a reagent of formula (5) or (6) is from about 1:1 to about 1:10. In some embodiments, the ratio of a polymer to a reagent of formula (5) or (6) is about 2:1. In some embodiments, conditions that facilitate formation of a monoderivatized product include the addition of an activating reagent. An activating reagent is then added to the mixture of the polymer and the reagent of formula (4) or (5). An activating reagent can be any group suitable to initiate coupling of the polymer and the reagent of formula (4). Suitable activating reagents include, for example, 1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT), 5-(benzylthio) 1H-tetrazole (BTT), Activator 42 (5-(3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole), 2 ethylthiotetrazole, 2-bezylthiotetrazole, 4,5-dicyanoimidazoleand 4,5-dicyanoimidazole (DCI). In some embodiments, an activating agent can be selected from pyridinium hydrochloride, pyridinium trifluoroacetage, and buffered carboxylic acids. In some embodiments, conditions that facilitate formation of a monoderivatized product include addition of an oxidizing agent to oxidize P- 3 to P- 5 . Suitable oxidizing agents and conditions can be readily determined by those of ordinary skill in the art. For example, an oxidant such as RuO4/NMO, Dess-Martin's reagent, DMSO/triflic anhydride, PDC, hydrogen peroxide, inorganic peroxides, nitric acid, nitrates, chlorite, chlorate, perchlorate, hypochlorite, peroxide, iodine, ozone, nitrous oxide, silver oxide, permanganate salts, hexavalent chromium compounds, chromic acid, dichromic acids, chromium trioxide, pyridinium chlorochromate, 54 WO 2013/186632 PCT/IB2013/001885 persulfuric acid, sulfoxides, sulfuric acid, Tollens' reagent, 2,2'-dipyridiyldisulfide (DPS), and osmium tetroxide may be used. In some embodiments, iodine can be used as an oxidizing agent. For example, a solution of iodine can be used and prepared by dissolving iodine in a mixture of pyridine, tetrahydrofuran and water. Elemental sulfur can be used for phosphite oxidation combined with formation of sulfurized product. In some embodiments, other more soluble and more reactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide (Beaucage reagent), phenylacetyl disulfide (PADS) or dimethylthiuram (DTD) can be used. Alternatively, peroxides exemplified by t-butyl hydrogen peroxide or m-chlorobenzoyl peroxide may be used for p 3 to p 5 oxidation. In some embodiments, an oxidizing reagent is selected from a group consisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide, acetone peroxide, sulfur, and thiuram disulfide. In some embodiments, R and/or R 1 is a protecting group or a hydrophobic separation handle. In some embodiments, the method can further include purifying the monoderivatized compound using chromatography (e.g., reverse phase chromatography). To the monoderivatized product, a reagent of formula (4) or formula (5) is added under conditions that facilitate the conversion of the monoderivatized product to a compound of formula (2). In some embodiments, the reagent is different from the reagent used to prepare the monoderivatized product. In some embodiments, the reagent is the same as that used to prepare the monoderivatized product. In some embodiments, the conditions are such that conversion of the monoderivatized product to the compound of formula (2) is quantitative. In some embodiments, conditions that facilitate formation of a compound of formula (2) include the addition of an activating reagent. An activating reagent is then added to the mixture of the monoderivatized product and the reagent of formula (5) or (6). An activating reagent can be any group suitable to initiate coupling of the polymer and the reagent of formula (5) or (6). Suitable activating reagents include, for example, 1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT), 5 -(benzylthio)- 1 H-tetrazole (BTT), Activator 42 (5 -(3,5 -bis(trifluoromethyl)phenyl)- 1 H tetrazole), 2-ethylthiotetrazole, 2-bezylthiotetrazole, 4,5-dicyanoimidazoleand 4,5 55 WO 2013/186632 PCT/IB2013/001885 dicyanoimidazole (DCI). In some embodiments, an activating agent can be selected from pyridinium hydrochloride, pyridinium trifluoroacetage, and buffered carboxylic acids. In some embodiments, conditions that facilitate formation of a compound of formula (2) include addition of an oxidizing agent to oxidize p 3 to p 5 . Suitable oxidizing agents and conditions can be readily determined by those of ordinary skill in the art. For example, an oxidant such as RuO 4 /NMO, Dess-Martin's reagent, DMSO/triflic anhydride, PDC, hydrogen peroxide, inorganic peroxides, nitric acid, nitrates, chlorite, chlorate, perchlorate, hypochlorite, peroxide, iodine, ozone, nitrous oxide, silver oxide, permanganate salts, hexavalent chromium compounds, chromic acid, dichromic acids, chromium trioxide, pyridinium chlorochromate, persulfuric acid, sulfoxides, sulfuric acid, Tollens' reagent, 2,2'-dipyridiyldisulfide (DPS), and osmium tetroxide may be used. In some embodiments, iodine can be used as an oxidizing agent. For example, a solution of iodine can be used and prepared by dissolving iodine in a mixture of pyridine, tetrahydrofuran and water. Elemental sulfur can be used for phosphite oxidation combined with formation of sulfurized product. In some embodiments, other more soluble and more reactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide (Beaucage reagent), phenylacetyl disulfide (PADS) or dimethylthiuram (DTD) can be used. Alternatively, peroxides exemplified by t-butyl hydrogen peroxide or m-chlorobenzoyl peroxide may be used for p 3 to p 5 oxidation. In some embodiments, an oxidizing reagent is selected from a group consisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide, acetone peroxide, sulfur, and thiuram disulfide. In some embodiments, R and/or R 1 is a protecting group or a hydrophobic separation handle. In some embodiments, the method can further include purifying the monoderivatized compound using chromatography (e.g., reverse phase chromatography). In some embodiments, the method further includes removal of one or more of the protecting groups. In some embodiments, the method further includes removal of one or more of the hydrophobic separation handles. For the methods provided above, the deprotection may involve, for example, either sequential or one-pot deprotection of certain protecting groups. Suitable reagents and conditions for the deprotection can be readily determined by those of ordinary skill in the art. 56 WO 2013/186632 PCT/IB2013/001885 For example, deprotection may be achieved upon treatment of the protected compound under conditions so that hydroxyl protecting groups, such as acetate, isopropylidine, benzylidine, trityl, and pixyl protecting groups, are removed from the protected compound. The acetate group can be cleaved under mild conditions, for example, by diluted solution of ammonia or by solution of potassium carbonate. The benzylidene and isopropylidene groups can be cleaved by hydrogenation or using acidic hydrolysis as reported by R.M. Hann et al., J. Am. Chem. Soc., 72, 561 (1950). In yet another example, the deprotection can be performed so that amino protecting groups, such as 9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz) protecting groups are cleaved from the protected compound. 9 fluorenylmethyl carbamate (FMOC) can be removed under mild conditions with an amine base (e.g., piperidine) to afford the free amine and dibenzofulvene, as described by E. Atherton et al., "The Fluorenylmethoxycarbonyl Amino Protecting Group," in The Peptides, S. Udenfriend and J. Meienhofer, Academic Press, New York, 1987, p. 1. t-butyl carbamate (Boc) can be removed, as reported by G.L. Stahl et al., J. Org. Chem., 43, 2285 (1978), under acidic conditions (e.g., 3 M HCl in EtOAc). Hydrogenation can be used to cleave the carboxybenzyl carbamate (cbz) protecting group as described by J. Meienhofer et al., Tetrahedron Lett., 29, 2983 (1988). In some embodiments, deprotection may be performed under anaerobic conditions. The deprotection may also be performed at ambient temperature or at temperatures of from about 20 60 0 C (e.g., 25, 30, 35, 40, 45, 50, or 55 0 C). In some embodiments, the method can also include isolating the compound by precipitation or crystallization. Compounds offormula (3) Also provided herein is a compound of formula (3): 57 WO 2013/186632 PCT/IB2013/001885 E R polymer O-P-Z 1 -L-M-R z2 K-G linking group or a salt form thereof, wherein: polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer backbone, wherein M 2 and the phosphonate-derived functional group are bonded at a different terminus of said polymer; E and El are independently 0 or S; K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selected from the group consisting of hydrogen, alkoxy and a hydrophobic separation handle; ZI and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; M is selected from a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof or a group reactive with a biologically active molecule;
M
2 is selected from 0, S or NH; and R is absent, a protecting group, a hydrophobic separation handle, or an activating group;
R
2 is hydrogen or a protecting group; wherein when M is a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof, then R is a protecting group or a hydrophobic separation handle; and 58 WO 2013/186632 PCT/IB2013/001885 wherein when M is a group reactive with a biologically active molecule or a derivative thereof, R is absent, hydrogen, or an activating group; and A polymer can be, for example, poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly (* -hydroxy acid), poly(vinyl alcohol), polyoxazoline, or a copolymer thereof. Such polyalkylene glycols, include, but are not limited to, polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have average molecular weights between about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, between about 500 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 500 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, and 500 Da. In some embodiments, the molecular weight of the polymer is between about 500 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 500 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the polymer is a poly(ethylene glycol) polymer. The molecular weight of the PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the n PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the 59 WO 2013/186632 PCT/IB2013/001885 molecular weight of the PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the PEG is between about 5,000 Da and about 20,000 Da. In some embodiments, E is oxygen. In some embodiments, E is sulfur. In some embodiments, K is a linear or branched alkylene. For example, K can be selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene. In some embodiments, K can be an alkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, K can be a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, K is selected from the group consisting of -(CH 2 )n 1 - and -((CH 2 )n-O-(CH 2 )m)p-, wherein n is an integer from I to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, Ito 10, ito 6, ito 6, ito 2, ito 3, ito 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, Ito 18, Ito 15, Ito 12, Ito 10, 1 to6, to2, to3, 1 to4,2to5,5to50, 10to50, 15to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently an integer from Ito 10(e.g., I to8, I to6, ito5, ito3,2to 10,4to 10,6to 10,2to8,and3to6). In some embodiments, G is a hydrophobic separation handle. For example, G can be a substituted or unsubstituted trityloxy group. In some embodiments, G is selected from the group consisting of monoalkoxy substituted trityloxy group or dialkoxy substituted trityloxy group. In some embodiments, one of Z' and Z 2 is NH and the other is 0. For example, Z' is 0 and Z2 is NH; Z is NH and Z 2 is 0. In some embodiments, both Z' and Z2 are 0. In some embodiments, L is a linear or branched alkyl. For example, L can be selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene. In some embodiments, L can be an alkyleneoxyalkylene or an oligomeric alkyleneoxyalkylene. For example, L can be a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. In some embodiments, L is selected from the group consisting of -(CH 2 )n- and -((CH 2 )n-O-(CH 2 )m)p-, wherein n is an integer from I to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12, Ito 10, ito 6, ito 6, ito 2, ito 3, ito 4, 2 to 50, 5 to 50, 10 to 50, 15 to 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), m is an integer from 0 to 50 (e.g., I to 40,1 to 30, 1 to 25, 1 to 20, Ito 18, Ito 15, Ito 12, Ito 10, 1 to6, I to2, I to3, I to4,2to5O,5to5O, 10to50, 15to 60 WO 2013/186632 PCT/IB2013/001885 50, 25 to 50, 5 to 15, 2 to 12, 20 to 30, and 6 to 18), and each p is independently an integer from Ito 10(e.g., 1 to8, 1 to6, Ito5, 1to3,2to 10,4to 10,6to 10,2to8,and3to6). In some embodiments, L is a substituted or unsubstituted arylene. For example, L can be a structure with the formula: Wr wherein W is a substituent and r is an integer from 0 to 4. For example, W can be selected from the group consisting of: halo, C 1 -Cio alkyl, C 2 -Cio alkenyl, C 2 -Cio alkynyl, C 1 -Cio alkoxy, C 5
-C
12 aralkyl, C 3
-C
12 cycloalkyl, C 4
-C
12 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C 2
-C
12 alkoxyalkyl, C 5
-C
12 alkoxyaryl, C 5
-C
12 aryloxyalkyl, C 7
-C
12 oxyaryl, C1-C 6 alkylsulfinyl, CI-Cio alkylsulfonyl, -(CH 2 )m-0-(C 1 -Cio alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO 2 , -CN, -NR 9
C(O)-(C
1
-C
1 O alkyl), C(O)-(C 1 -Cio alkyl), C 2 -Cio alkthioalkyl, -C(O)O-(C 1 -Cio alkyl), -OH, -SO 2 , =S, COOH, -NR9 2 , carbonyl, -C(O)-(C 1 -Cio alkyl)-CF 3 , -C(O)-CF 3 , -C(O)NR 9 2 , -(C 1 -Cio aryl)-S-(C 6 -Cio aryl), -C(O)-(C 6 -Cio aryl), -(CH 2 )m-0-(CH 2 )m-0-(C 1
-C
1 O alkyl) wherein each m is from 1 to 8, -C(O)NR 9 2 , -C(S)NR 9 2 , -S0 2
NR
9 2 , -NR 9
C(O)NR
9 2 , NR 9
C(S)NR
9 2 , salts thereof, and the like. Each R9 group in the preceding list independently includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkylaryl. In some embodiments, W is R 1 as described above. Non-limiting examples of L include: 61 WO 2013/186632 PCT/IB2013/001885 r r Wr Wwr L can also be a divalent radical of a nucleoside. For example, L can be a divalent radical of a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, 5 methyluridine, thymidine, uridine, deoxyuridine, cytidine, and deoxycytidine. A group reactive with a biologically active molecule or a derivative thereof can be selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide. In some embodiments, the group is protected or further reacted with a group R as shown in the structure of formula (3). The point of attachment of such a group is well understood by those of skill in the art. In some embodiments, R is absent. In some embodiments, R 2 is hydrogen. In some embodiments, R and/or R 2 is a protecting group. For this purpose, R and/or R 2 may include any suitable protecting group based on the group to be protected. For example, R and/or R 2 may include any suitable hydroxyl functional group including, but not limited to, ether, ester, carbonate, or sulfonate protecting groups. In particular, the ether protecting group may include benzyloxymethyl (BOM), methylthiomethyl (MTM), phenylthiomethyl (PTM), , cyanoethyl, 2,2-dichloro- 1,1 difluoroethyl, 2-chloroethyl, 2-bromoethyl, tetrahydropyranyl (THP), , phenacyl, 4 bromophenacyl, , allyl, propargyl, , t-butyl, benzyl, 2,6-dimethylbenzyl, 4-methoxybenzyl 62 WO 2013/186632 PCT/IB2013/001885 (MPM-OAr), o-nitrobenzyl, 2,6-dichlorobenzyl, 3,4-dichlorobenzyl, 4 (dimethylamino)carbonylbenzyl, 4-methylsulfinylbenzyl (Msib), 9-anthrylemethyl, 4-picolyl, heptafluoro-p-tolyl, tetrafluoro-4-pyridyl, trimethylsilyl (TMS), , and protecting groups. The ester protecting group may include acetoxy (OAc), aryl formate, acetate, levulinate, pivaloate, benzoate, and 9-fluoroenecarboxylate. In one embodiment, the ester protecting group is an acetoxy group. The carbonate protecting group may include aryl methyl carbonate, 1 -adamantyl carbonate (Adoc-OAr), t-butyl carbonate (BOC-OAr), 4-methylsulfinylbenzyl carbonate (Msz OAr), 2,4-dimethylpent-3-yl carbonate (Doc-OAr), aryl 2,2,2-trichloroethyl carbonate,. The sulfonate protecting groups may include aryl methanesulfonate, aryl toluenesulfonate, and aryl 2-formylbenzenesulfonate. In some embodiments, R may include any suitable amino protecting group, including, but not limited to, carbamate, amide, N-alkyl, or N-aryl-derived protecting groups. In particular, the carbamate protecting group may include, for example, 9 fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), carboxybenzyl carbamate (cbz), methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7 dibromo)fluorenylmethyl carbamate, 17-tetrabenzol[a,c,g,i]fluorenylmethyl carbamate (Tbfmoc), 2-chloro-3-indenylmethyl carbamate (Climoc), 2,7-di-t-butyl[9-(10,10-dioxo 10,10,10,1 0-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 1,1 dioxobenzo[b]thiophene-2-ylmethyl carbamate (Bsmoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1,1-dimethyl-2-haloethyl carbamate, 1,1 -dimethyl-2,2-dibromoethyl carbamate (DB-t-boc), 1,1 -dimethyl-2,2,2 trichloroethyl carbamate (TCBoc), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di t-butylphenyl)- 1 -methylethyl carbamate (t-Bumeoc), N-2-pivaloylamino)- 1,1 -dimethylethyl carbamate, 2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate (NpSSPeoc), 2-(N,N dicycloheylcarboxamido)ethyl carbamate, 1-adamanyl carbamate (1-Adoc), cinyl carbamate (Voc), 1-isopropylallyl carbamate (Ipaoc), 4-nicrocinnamyl carbamate (Noc), 3-(3'pyridyl)prop 2-enyl carbamate (Paloc), 8-quinolyl carbamate, alkyldithio carbamate, p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate (Pnz), p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 63 WO 2013/186632 PCT/IB2013/001885 diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4 methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2 phosphonioethyl carbamate (Peoc), 1,1-dimethyl-2-cyanoethyl carbamate, 2-(4-nitrophenyl)ethyl carbamate, 4-phenylacetoxybenzyl carbamate (PhAcOZ), and m-chloro-p-acyloxybenzyl carbamate. In some embodiments, the carbamate protecting group is chosen from 9 fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz). The amide protecting group may include, for example, acetamide, phenylacetamide, 3 phenylpropanamide, pent-4-enamide, picolinamide, 3-pyridylcarboxamide, benzamide, p phenylbenzamide, 2-methyl-2-(o-phenylazophenoxy)propanamide), 4-chlorobutanamide, acetoacetamide, 3-(p-hydroxyphenyl)propanamide), and (N' dithiobenzyloxycarbonylamino)acetamide. Examples of suitable protecting groups also include tert-butyl, benzyl, 4-methoxybenzyl, benzyloxymethyl, phenacyl, allyl, trimethylsilyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acetal and ketal derivatives. In some embodiments, R is selected from trityls, substituted trityls (e.g., monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), trimethoxytrityl (TMTr), 2 chlorotrityl (ClTr) and p-bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, for example, U.S. Publication No. 2007/0276139). In some embodiments, R is selected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal. In some embodiments, R is a hydrophobic separation handle. A hydrophobic separation handle is as described herein. In some embodiments, the hydrophobic separation handle is also a protecting group as described herein. In some embodiments, at least one of R and G is a hydrophobic separation handle. In some embodiments, only one of R and G is a hydrophobic separation handle (e.g., a trityl group) as provided herein. For example, if R is a hydrophobic separation handle, then G is hydrogen or an alkoxy. In some embodiments, R is a protecting group and G is hydrogen or an alkoxy. In some embodiments, R is absent and G is a trityloxy group. In some embodiments, 64 WO 2013/186632 PCT/IB2013/001885 the hydrophobic separation handle is a substituted or unsubstituted trityl or trityloxy group. For example, only one of R and G is a substituted or unsubstituted trityl or trityloxy group. In some embodiments, R 2 is absent or is selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl, and methyl. A compound of formula (3) can be prepared, for example, by contacting a monosubstituted polymer comprising a linear, water-soluble, non-peptidic and non-nucleotidic polymer backbone bonded at the first terminus with the functional group M 2
-R
2 , with a reagent of formula (5): Rs5
N--P-Z
1 -L-M--R R6/ I Z2 K -G wherein:
R
5 and R 6 independently from each other represent C1-C 6 -alkyl or R 5 and R6 jointly form a 5- or 6-membered ring with the N to which they are bonded; under conditions facilitating the conversion of the monosubstituted polymer to a compound of formula (3). In some embodiments, R 5 and R6 are independently a C1-C 6 -alkyl. For example, R 5 and R6 can be independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and hexyl. In some embodiments, R 5 and R 6 are isopropyl. In some embodiments, R 5 and R6 jointly form a 5- or 6-membered ring with the N to which they are bonded. For example, R 5 and R6 jointly form a pyrrolidine, pyrroline, imidazoline, pyrazolidine, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyridyl, pyrazinyl, pyrimidinyl, particularly 2- and 4-pyrimidinyl, pyridazinyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, and pyrazolyl, particularly 3- and 5-pyrazolyl. In some embodiments, R 5 and R6 jointly form a morpholine ring. The ratio of a monosubstituted polymer to a reagent of formula (5) can range from about 1:10 to about 10:1 (e.g., about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:81 about 1:9, about 2:8, about 3:7, about 4:6 about 5:10, and about 4:8). In some embodiments, the ratio 65 WO 2013/186632 PCT/IB2013/001885 of a polymer to a reagent of formula (5) is from about 1:1 to about 1:10. In some embodiments, the ratio of a polymer to a reagent of formula (5) is about 2:1. In some embodiments, the conversion of a monosubstituted polymer to a compound of formula (3) is quantitative. In some embodiments, conditions that facilitate formation of a compound of formula (3) include the addition of an activating reagent. An activating reagent is then added to the mixture of the monoderivatized product and the reagent of formula (5) or (6). An activating reagent can be any group suitable to initiate coupling of the polymer and the reagent of formula (5) or (6). Suitable activating reagents include, for example, 1H-tetrazole, 5-(ethylthio)-1H-tetrazole (ETT), 5 -(benzylthio)- 1 H-tetrazole (BTT), Activator 42 (5 -(3,5 -bis(trifluoromethyl)phenyl)- 1 H tetrazole), 2-ethylthiotetrazole, 2-bezylthiotetrazole, 4,5-dicyanoimidazole and 4,5 dicyanoimidazole (DCI). In some embodiments, an activating agent can be selected from pyridinium hydrochloride, pyridinium trifluoroacetage, and buffered carboxylic acids. In some embodiments, conditions that facilitate formation of a compound of formula (3) include addition of an oxidizing agent to oxidize p 3 to p 5 . Suitable oxidizing agents and conditions can be readily determined by those of ordinary skill in the art. For example, an oxidant such as RuO 4 /NMO, Dess-Martin's reagent, DMSO/triflic anhydride, PDC, hydrogen peroxide, inorganic peroxides, nitric acid, nitrates, chlorite, chlorate, perchlorate, hypochlorite, peroxide, iodine, ozone, nitrous oxide, silver oxide, permanganate salts, hexavalent chromium compounds, chromic acid, dichromic acids, chromium trioxide, pyridinium chlorochromate, persulfuric acid, sulfoxides, sulfuric acid, Tollens' reagent, 2,2'-dipyridiyldisulfide (DPS), and osmium tetroxide may be used. In some embodiments, iodine can be used as an oxidizing agent. For example, a solution of iodine can be used and prepared by dissolving iodine in a mixture of pyridine, tetrahydrofuran and water. Elemental sulfur can be used for phosphite oxidation combined with formation of sulfurized product. In some embodiments, other more soluble and more reactive reagents, such as 3H-1,2-benzothiazol-3-one 1,1-dioxide (Beaucage reagent), phenylacetyl disulfide (PADS) or dimethylthiuram (DTD) can be used. Alternatively, peroxides exemplified by t-butyl hydrogen peroxide or m-chlorobenzoyl peroxide may be used for p 3 to p 5 oxidation. 66 WO 2013/186632 PCT/IB2013/001885 In some embodiments, an oxidizing reagent is selected from a group consisting of: iodine, hydrogen peroxide, t-butyl hydrogen peroxide, acetone peroxide, sulfur, and thiuram disulfide. In some embodiments, R and/or R 1 is a protecting group or a hydrophobic separation handle. In some embodiments, the method can further include purifying the monoderivatized compound using chromatography (e.g., reverse phase chromatography). In some embodiments, the method further includes removal of one or more of the protecting groups. In some embodiments, the method further includes removal of one or more of the hydrophobic separation handles. For the methods provided above, the deprotection may involve, for example, either sequential or one-pot deprotection of certain protecting groups. Suitable reagents and conditions for the deprotection can be readily determined by those of ordinary skill in the art. For example, deprotection may be achieved upon treatment of the protected compound under conditions so that hydroxyl protecting groups, such as acetate, isopropylidine, benzylidine, trityl, and pixyl protecting groups, are removed from the protected compound. The acetate group can be cleaved under mild conditions, for example, by diluted solution of ammonia or by solution of potassium carbonate. The benzylidene and isopropylidene groups can be cleaved by hydrogenation or using acidic hydrolysis as reported by R.M. Hann et al., J. Am. Chem. Soc., 72, 561 (1950). In yet another example, the deprotection can be performed so that amino protecting groups, such as 9-fluorenylmethyl carbamate (Fmoc), t-butyl carbamate (Boc), and carboxybenzyl carbamate (cbz) protecting groups are cleaved from the protected compound. 9-fluorenylmethyl carbamate (FMOC) can be removed under mild conditions with an amine base (e.g., piperidine) to afford the free amine and dibenzofulvene, as described by E. Atherton et al., "The Fluorenylmethoxycarbonyl Amino Protecting Group," in The Peptides, S. Udenfriend and J. Meienhofer, Academic Press, New York, 1987, p. 1. t-butyl carbamate (Boc) can be removed, as reported by G.L. Stahl et al., J. Org. Chem., 43, 2285 (1978), under acidic conditions (e.g., 3 M HCl in EtOAc). Hydrogenation can be used to cleave the carboxybenzyl carbamate (cbz) protecting group as described by J. Meienhofer et al., Tetrahedron Lett., 29, 2983 (1988). 67 WO 2013/186632 PCT/IB2013/001885 In some embodiments, deprotection may be performed under anaerobic conditions. The deprotection may also be performed at ambient temperature or at temperatures of from about 20 60 0 C (e.g., 25, 30, 35, 40, 45, 50, or 55 0 C). In some embodiments, the method can also include isolating the compound of formula (3) by precipitation or crystallization. Non-limiting examples of linking groups for use in the compounds provided herein include: MMTr NH 0 MMTr O-NH 0 0 O-CITr O-CITr 0 0 - F -O-P-O - -O-P-O 68 33 68 WO 2013/186632 PCT/IB2013/001885 DMTr HN S 0 NH 0
---
0 4 o HN-FMOC DMTr NH 0 0 0 0 HN-FM0C DMTr N H -0 0 - -- 0 0 69 WO 2013/186632 PCT/IB2013/001885 0CH 3 0 O 00 0
O-CH
3 0 ~ \ / O-CH 3 0 - O-CH 3
O-CH
3 - -O-P-O 70 WO 2013/186632 PCT/IB2013/001885 0 O-N O O 0 Tr MMTr NH 0 0 O-CITr o0 0 0O 00 -NH HN S 0 Tr O NH 0 - -O-P -O 15 71 WO 2013/186632 PCT/IB2013/001885 o HN-FMOC DMTr NH 0 0 OT
OCH
3 0 0 0 0 CI
O-CH
3 0 O \ / O-CH 3 O-P-O 72 WO 2013/186632 PCT/IB2013/001885 0 O-N 0 00 0 Tr MMTr NH S O-CITr 0 DMTr HN S 0 NH S 35 73 WO 2013/186632 PCT/IB2013/001885 o HN-FMOC DMTr NH 0 S 0 00 O'C H 3 0 0 S 0 Tr
O-CH
3 0 \/ O-CH 3 - 1 O- -7 74 WO 2013/186632 PCT/IB2013/001885 O-N S 0 Tr O-N S 0o Tr MMTr NH 0 75 WO 2013/186632 PCT/IB2013/001885 N H HN S 0 Tr O NH S o HN-FMOC DMTr NH 0 S\ / O
CH
3 0 0 0 S - -O- J-O Tr 76 WO 2013/186632 PCT/IB2013/001885 CI
O-CH
3
O
O-CH
3 --- P-0 MMTr NH 0 -- NH 0 O-CITr o 0 P-NH 77 WO 2013/186632 PCT/IB2013/001885 0 DMTr N HN S 0 NH 0 SO-P-NH o HN-FMOC DMTr NH 0 0 - -O-P-NH 0 78 WO 2013/186632 PCT/IB2013/001885 OCH3 0 0 O 0 O- -N H OTr
O-CH
3 0 0\/
O-CH
3 -0-P-NH 79 WO 2013/186632 PCT/IB2013/001885 MMTr NH 0. 0 +O-P-NH IO P-NHN NH HN S 0 Tr 0 NH 0 -F--NH 0' o HN-FMOC DMTr NH O 0 -0-P-NH 80 WO 2013/186632 PCT/1B2013/001885 0H 0 0 00 IC ___ O-CH 3 o
O-CH
3 -0--P-NH 81 WO 2013/186632 PCT/IB2013/001885 MMTr NH S II O O-CITr S 0 P-NH 0 DMTr N HN S 0 NH S P-NH 0 82 WO 2013/186632 PCT/IB2013/001885 0 HN-FMOC DMTr NH 0 S - -O-P-NH O O 0 OCH3 00 0 O S - -O-P-NH O 0 83 WO 2013/186632 PCT/IB2013/001885
O-CH
3
O-CH
3 -- P-NH 0 0 MMTr NH 0. S + -P-NH I P-NHN 0O NH HN S 0 Tr 0 NH S -- O-F-NH 08 84 WO 2013/186632 PCT/IB2013/001885 0 HN-FMOC DMTr NH 0 S - -O-P-NH 0 O 0 OCH3 0 0 O OTr 85 WO 2013/186632 PCT/IB2013/001885 CI
O-CH
3
O
O-CH
3 -- P-NH MMTr NH 0 OP-O O-CITr 0 0 HN 8Tr 86 WO 2013/186632 PCT/IB2013/001885 0 DMTr N HN S 0 NH 0 HN 5 o HN-FMOC DMTr NH 0 0 - O- -O NH 0 87 WO 2013/186632 PCT/IB2013/001885
O-CH
3
O-CH
3 N H 01 OCH3 0 o 0 0 NH OTr 88 WO 2013/186632 PCT/IB2013/001885 MMTr NH 0. 0
--
O-P-O NH 0 NH HN S 0 Tr 0 NH 0 HN +0-P-0 5 o HN-FMOC DMTr NH 0 0 NH 0 89 WO 2013/186632 PCT/IB2013/001885 OCH3 0 O O 0 0 NH OTr CI _ O-CH 3
O-CH
3 NH 090 90 WO 2013/186632 PCT/IB2013/001885 MMTr NH S +OP-O O-CITr S 0 0O 3 DMTr N HN S 0 NH S HN 5 91 WO 2013/186632 PCT/IB2013/001885 0 HN-FMOC DMTr NH 0 S NH O +~ O1 - O- -O NH OTr 92 WO 2013/186632 PCT/IB2013/001885
O-CH
3
O-CH
3 N H 0 MMTr -NH - O-P-O NH-N NHH HN S P-O 0 Tr O NH S -- O- -O HN 93 WO 2013/186632 PCT/IB2013/001885 0 HN-FMOC DMTr NH 0 S NH 0 +~ O1 O-P-0O 00 0 NH OTr 94 WO 2013/186632 PCT/IB2013/001885 CI -_
O-CH
3
O-CH
3 --- P-0 NH Provided herein is a new type of functional, water-soluble polymer, not belonging to the classes of polypeptides or nucleotides and containing the structure of formula (1) that can be conjugated to a biologically active molecule or a derivative thereof: E R1
A-O-P-Z
1 -L-M-R Z2 K I G In this schematic picture of such a modified polymer, A is the point of bonding to the terminus of the polymer backbone, E is an oxygen or sulfur atom, K is selected from the group consisting of alkylene, alkyleneoxyalkylene, or an oligomeric form of alkyleneoxyalkylene, G is hydrogen or is selected from the group consisting of an alkoxy and a hydrophobic separation handle, Z' and Z 2 can be oxygen or nitrogen, in such way that both Z' and Z 2 may be oxygen, but when Z' is NH then Z 2 is oxygen, and when Z 2 is NH then Z' is oxygen, L is selected from the group consisting of a divalent radical of a nucleoside, linear alkylene, branched alkylene, alkyleneoxyalkylene, oligomeric form of alkyleneoxyalkylene, arylene, and substituted arylene, M is a protected group that when deprotected is reactive with a biologically active molecule or a derivative thereof or is a group reactive with a biologically active molecule or a derivative 95 WO 2013/186632 PCT/IB2013/001885 thereof, R is a protecting group, activating group, hydrogen or absent. Thus the L-M-R fragment is linked to a terminus of the polymer via a phosphotriester, thiophosphotriester or amidophosphotriester group. One characteristic of a compound provided herein is that the functional group M, via the group L, is connected to the chain of the polymer via a phosphotriester group or amido phosphodiester, known also as phosphoramidate group. In addition, a derivatized polymer may exist both in an oxy and a thio form. Non-limiting examples of such groups include: O s polymer-O-P-O-L-M-R polymer-O-P-O-L-M-R K-G K-G 0 O 11H 11yerOPO-- polymer- -O-P-N-L-M--R polymer- -- P-0-L-M-R K-G K-G An important class of polymers provided herein, polyethylene glycols (PEG), were previously used in the synthesis of phosphoramidites of the formula: DMTr-O-PEG-O P(OCE)N(iPr) 2 . These compounds were used for direct coupling of PEG molecules to synthetic nucleic acids or to a surface of a solid phase. In all reactions, the reactive phosphoramidite group was present at the terminal of the polymer. A polymer substituted with a phosphoramidite group is, however, not a subject of the present disclosure, as the phosphorous atom in the phosphoramidite group is the part of the reactive functionality and not a part of the linker as in the compounds provided herein. This formal distinction can have a deeper chemical importance. A phosphoramidite group can be designed to work in a completely water-free environment. Upon activation with certain protonating agents/activators a phosphoramidite group can become extremely reactive, and in the presence of water, this reactive function can decompose instantaneously, making this function inappropriate for conjugation to a biologically active molecule or a derivative thereof in aqueous solution. Additionally, published PEG phosphoramidites can contain a specially designed, labile protecting group adjacent to the phosphorous atom to convert the intermediate phosphotriester to 96 WO 2013/186632 PCT/IB2013/001885 a phosphodiester. The present document is based, at least in part, on the observation that phosphodiesters are unstable in aqueous biological media such as blood, plasma or cellular extracts, due to the presence of phosphatases and phosphodiesterases. This can preclude phosphodiester linkages as a linking group within a structure of a conjugate, as long as it is aimed for use in biological media. This document, contrary to the existing literature, and contrary to all normal procedures, recommends keeping the phosphate in the form of a phosphotriester or in the form of its amide in order to gain stability of the linking group/conjugate. Phosphotriester bonds are very rare in nature, existing in most of the cases as cyclic products of RNA transformations. As non-charged variants of nucleotides they gained some attention from those who hoped that it could be possible to use this form of nucleotides as predrugs, which would be transformed in vivo to the active phosphodiester forms. However, acyclic phosphotriesters, lacking specially designed internal features facilitating deprotection, were found completely stable, both chemically at the physiological pH range, and enzymatically in the presence of the most active phosphate hydrolytic enzymes: McGuigan et al., Nucl. Acids Res. 1989, 17 (15), 6065-6075; Hecker and Erion, J. Med. Chem. 2008, 51, 2328-2345; Conrad et al. Chem. Bio. Interactions 1986, 60, 57-65; and Fidanza et al., Methods in Molecular Biology 1994, 26, 121-143. The proposed way of linking a functional group to the polymeric molecule can include combining chemistry typical for nucleic acids with chemistry of polymers and their conjugates. Moreover, this combination can be performed in a way to yield a product with distinctly better characteristics than if this combination of chemistries would proceed following the standard path. This document also provides methods and materials for introducing of a useful separation handle on the derivatized polymer. This separation handle can be introduced simultaneously with the functional group, so the presence of the separation handle becomes an indicator of successful introduction of the reactive group. If chromatographic properties of a particular separation handle are properly chosen, it is possible to discriminate between non-derivatized, monoderivatized and multiderivatized (e.g., bis-derivatized) polymers. Most of the separation handles used herein introduce hydrophobic properties to the polymer, as the preferred method for 97 WO 2013/186632 PCT/IB2013/001885 separation of the modified polymers is based on reverse-phase analytical and preparative chromatography. The choice of a proper separation handle comes from consideration of several practical aspects such as: a) The hydrophobic separation handle can be removed from the polymer after purification in order to liberate the group reactive with a biologically active molecule and to avoid uncontrolled hydrophobic interactions within the conjugate-or more generally to avoid uncontrolled hydrophobic interactions during the interaction of the conjugate with the biological environment. This can preclude work with analogues of mPEG which contain a long hydrophobic alkyl ether chain instead of a methoxy group at one of the polymer termini. For the same reason, protection of an amino group as long chain fatty acid amides is not practical, as this group can be removed only under very extreme conditions. b) As most of polymers and functional groups lack chromophoric properties, the chromatographic separation of polymers can be difficult. It is, therefore, advantageous if the separation handle introduces additionally some chromophore properties to the polymer. This can make protection of a terminal hydroxyl or thiol group by means of a long chain aliphatic fatty acid ester less interesting. c) Chemical stability of the hydrophobic separation handle can be easy to control depending on an actual situation. This aspect is related mostly to the stability of other functional groups present in the derivatized polymer. d) Chromatographic properties of the hydrophobic separation handle, and hence the properties of the derivatized polymer, can be easy to control by chemical modification of the hydrophobic separation handle. e) Since even relatively stable phosphotriester bonds are slightly labile under high pH conditions, it can be preferred to avoid hydrophobic separation handles that can only be removed under such conditions. Examples of hydrophobic separation handles that fulfill all these criteria belong to the group of acid labile protecting groups and are known as trityls, substituted trityls (e.g., monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), trimethoxytrityl (TMTr), and p bromophenacyloxytrityl (BPTr), pixyls and substituted pixyls (see, for example, U.S. Publication No. 2007/0276139). They are all acid labile, with distinct UV chromophore properties. Their 98 WO 2013/186632 PCT/IB2013/001885 acid stability can be easily controlled by the presence of different electron donating or electron withdrawing groups. Introduction of alkoxy chains of different length to the trityl structure is a convenient method for modification of their hydrophobic properties. Most of the Examples presented herein utilize, therefore, trityl groups both for protection of reactive functions and for introduction of an efficient separation handle. This should not be seen as a limitation of this disclosure, as other groups, even those which do not fulfill all the above criteria, may be useful in the present methodology as hydrophobic separation handles. Thus, a general description of a potential hydrophobic separation handle provided herein is: a hydrophobic group that withstands the presence of trivalent phosphorus present in a phosphoramidite or in an activated H phosphonate, and which can also be employed for chromatographic resolution of modified polymer from the unmodified starting material. With a few exceptions, there are few limits on the type and character of a functional group M. These exceptions appear in cases when the functional group is very sensitive to reducing conditions and becomes destroyed by the trivalent phosphoramidite group, with concomitant oxidation of phosphorus to the pentavalent oxidation state. The azido group is an example of such a reactive function that cannot be converted to the appropriate phosphoramidite. In fact, trivalent phosphorus of triphenylphosphine is used as an efficient reagent for conversion of an azido group to an amine. The activated dithio group, for example, as in a dithiopyridyl group, is another example that belongs to this category, although simple dithiols could be successfully converted to and delivered in the form of a phosphoramidites. Nevertheless, this disclosure presents also a variant of the above phosphoramidite method that omits the mentioned stability problem, and provides the ability to prepare polymers containing even an azido or activated dithio group. This method is known as H-phosphonate methodology followed by oxidation of p 3 by carbon tetrachloride/amine, and is similar to the phosphoramidite method in the sense that the incoming reagent contains reactive p 3 phosphorus, and the phosphorus atom is oxidized during the reaction process to its pentavalent stage. The H-phosphonate methodology will be described in more detail later on in this text. Many groups reactive with a biologically active molecule or a derivative thereof need to be protected in order to exist within the structure of phosphoramidite reagent. Examples include, 99 WO 2013/186632 PCT/IB2013/001885 without limitation, amino, aminoxy, hydrazo, hydroxyl, thio, certain fluorophores and carboxy groups. Some of these groups, like biotin, do not demand protection, but can be used in a protected form to obtain some additional effects. In some cases, trityl, substituted trityl, pixyl, and substituted pixyl can be used as protecting groups. One reason for this choice of a protecting group is the possibility for simultaneous introduction of a protecting group that also can be used as a hydrophobic separation handle in a reverse phase (RP) based chromatographic separation process. If separation is not demanded, like in the case where the polymer has only one reactive terminus (e.g., when using mPEG as a polymer backbone), and incorporation of a phosphoramidite may be forced to completion, any protecting group can be used for protection of the group reactive with a biologically active molecule. In particular, trifluoroacetamido and FMOC groups may be used for the protection of amino, aminoxy and hydrazo groups in such phosphoramidites. The use of phosphoramidites containing a protected group, that when unprotected is reactive with a biologically active molecule or a derivative thereof, having a hydrophobic protecting group for derivatization of polymers has a clear advantage over other methods. For example, the starting polymer does not have to be partially protected in order to obtain pure, monofunctionalized polymers. The methods work with fully unblocked polymers and improved yield is obtained using an excess of such a non-expensive polymer (e.g., non-derivatized PEG) over the amidite. One value of this method is in the fact that the formed mixture consisting of mono-, bis-, or multi-derivatized polymers can be efficiently separated from each other. Certain functional groups do not offer a straightforward possibility for introduction of a desired hydrophobic protecting group. To this group belong NHS-esters, most of the fluorophores, iodoacetamido and maleimido groups. Amidites containing these functional groups are not optimal for derivatization of diol-polymers, since the separation of the reaction mixtures can be impossible. In some cases, polymer diols can be easily converted to certain monoprotected derivatives, and this protection can offer the possibility for using chromatography to obtain pure, temporarily blocked monoderivatized polymers. Even here the separation can be based on hydrophobic interactions between a hydrophobic support and a polymer derivatized with a hydrophobic protecting group acting as a hydrophobic separation handle. Examples of handles 100 WO 2013/186632 PCT/IB2013/001885 used for this purpose include, without limitation, substituted or unsubstituted tritylated hydroxyls, tritylated thiols, and tritylated amines. Introduction of a trityloxy or pixyloxy group onto the terminus of a polymer is straightforward as these polymers usually contain free hydroxyl groups. Introduction of a tritylthio group requires activation of the hydroxyl group by its conversion to a mesylate, tosylate, or by substitution with a halogen. In some cases, this last alternative can be used, as its implementation in the form of a Velsmeier reaction can be economically attractive. The activated polymer can then react with tritylmercaptan as described by Conolly and Rider in Nucleic Acids Res. 1985, 13, (12), 4485-4502. A polymer substituted with a thiol group also can be obtained by any of the other described methodologies, and then the thio group can be selectively tritylated in an acidic environment, utilizing the much higher affinity of the thio group over hydroxyl to carbocations. There are two ways to obtain polymers protected on one site with a tritylated amine group. A process is described for direct alkylation of tritylamine with alkyl halides so this method could be used directly in analogy to the above alkylation of tritylmercaptan. On the other hand any appropriate method for partial amination like alkylation of the phthalimide, alkylation of the trifluoracetamide or a Mitsunobu procedure can be applied for preparation of a monoamino polymer. This starting material, even in unpurified form, can be used for obtaining the tritylated amino polymer in a two-step process, starting with silylation of all free hydroxyls in a pyridine solution. All mentioned tritylated or pixylated polymers can be preoperatively purified by RP chromatography to isolate the pure monosubstituted polymer. The described earlier mono-protected polymer derivatives, made by reacting a polymer with a selected phosphoramidite, offer an interesting alternative as a mono protected polymeric starting material for additional derivatization. Such a polymer can react next with a phosphoramidite to incorporate another functional group, so the final product can contain, for example, two separate phosphotriester linkages. Monomethoxy PEG (mPEG), or generally monoalkylated polymers, can be used for the same purpose. Methoxy PEG's are used to guarantee monofunctionalization of a polymer. The problem is that such polymers can be contaminated by an unknown and variable amount of bis hydroxyl (diol) polymer. In the case of PEG the existence of the diol form is a consequence of moisture present during the polymerization of the ethyleneoxide, and this can be hard to avoid on an industrial scale. Even the slightest amount of water can result in formation of a hydroxyl 101 WO 2013/186632 PCT/IB2013/001885 anion - an undesired starting point of polymerization. Thus mPEG is not an optimal polymer for preparation of pure monofunctionalized polymers. Once a polymer is properly monoprotected, however, its conversion to a reactive monofunctional derivative is straight-forward. Phosphoramidite chemistry allows incorporation of a reactive functionality with yield and speed that is beyond the competition of other chemical processes, including all sorts of hydroxyl alkylation reactions. Using the proper excess of these reagents, the reported yields exceed 98% for every step in a multistep process and are often nearly quantitative. In cases where only a single incorporation of a phosphoramidite takes place, this reaction can be expected to be quantitative. There exists also an interesting alternative process to the described above functionalization following the previously described monoprotection. Groups reactive with a biologically active molecule or a derivative thereof that lack the possibility of carrying a hydrophobic separation handle can be incorporated into a trisubstituted unit having the reactive group of choice, the hydrophobic separation handle and the phosphoramidite group. There are several such units or molecules that can be used as carriers (scaffolds) for the construction of such trisubstituted block reagents. One of the simplest molecules, due to the ease of the chemical manipulations, is the uridine-based scaffold described by Hovinen and Hakala in Organic Lett. 2001, 3(16) 2473-2476, where R denotes a reactive group and L is an aliphatic linker to the uridine ring. 102 WO 2013/186632 PCT/IB2013/001885 O R N DMTr-O N 0 P-N(iPr) 2 0iPr or DMTr-O R 0 ,P-N(iPr) 2 0 i Wr For instance, using reagents belonging to this category, it is possible to prepare, in a single reaction step, polymers covalently bonded to different fluorescent functionalities that can be isolated as a monosubstituted polymeric product. The chemistry of phosphoramidites is mostly associated with the chemistry of nucleic acids. The very high demands of this multistep process require rather large excess of incoming amidites, and efficient catalysts. Examples of activators includes, without limitation, 1H tetrazole, 5-(ethylthio)-1H-tetrazole (ETT), 5-(benzylthio)-1H-tetrazole (BTT), Activator 42 (5 (3,5-bis(trifluoromethyl)phenyl)-1H-tetrazole), and 4,5-dicyanoimidazole (DCI). Those are often expensive, and if applied to the synthesis of polymers, as in this disclosure, they would noticeably increase the price of the final reagent. However, as the chemistry in the present disclosure can have a single coupling step, and the quantitative yield of the reaction is not always necessary, it is possible to use other less expensive catalysts. The use of pyridinium 103 WO 2013/186632 PCT/IB2013/001885 hydrochloride or even buffered carboxylic acids for activation of phosphoramidites is described and proved, offering high reaction rate, albeit with minimally lower coupling yield. The choice of an oxidizing reagent is another factor for consideration. Iodine is the simplest alternative, as this reagent can be prepared by dissolving iodine in a mixture of pyridine, tetrahydrofuran and water. Elemental sulfur can be used for phosphite oxidation combined with formation of sulfurized product, but it can be poorly soluble in organic solvents which can be used in the procedures described herein. Other, better soluble and more reactive reagents, like 3H-1,2-benzothiazol-3-one 1,1-dioxide (Beaucage reagent), phenylacetyl disulfide (PADS) or dimethylthiuram (DTD) were developed for this purpose. Thiophosphotriester linkages do not offer very much advantage over the normal phosphotriester linkages, as the latter can already be sufficiently stable, but may offer additional possibilities, e.g. the ability of chemical cleavage of this linkage. Peroxides exemplified by t-butyl hydrogen peroxide or m-chlorobenzoyl peroxide can be used as alternatives for p 3 to p 5 oxidations. They are colorless, can work under water free conditions and are often applicable in situations where iodine promoted oxidations may lead to some unwanted side reactions. It was mentioned earlier that groups reactive with a biologically active molecule which do not tolerate a coexistence with the phosphoramidite group can be attached to the polymer by H-phosphonate chemistry. This method takes advantage of the fact that p 3 of the H-phosphonate is actually tetracoordinated, and as such this group can be more stable for oxidation than a phosphate triester or phosphoramidite. The particularly important point is that H-phosphonate does not interact with an azido group like most p 3 containing compounds. The use of this methodology for incorporation of the azido group can be, however, combined with the incorporation of a useful hydrophobic separation handle allowing for discrimination between the product and unreacted starting material. Application of a trifunctional reagent, similar to the mentioned uridine derivative, but using a H-phosphonate instead of a phosphoramidite group, could be an alternative. In this case, the activation of the starting H-phosphonate reagent and its reaction with the polymer can be followed by addition of an excess of an appropriate alcohol (e.g., isopropanol) to convert the formed H-phosphonate diester to the desired H-phosphonate triester. The Examples below describe a slightly different procedure. Here, the procedure of introducing an azido group and a hydrophobic separation handle has been divided into two steps, 104 WO 2013/186632 PCT/IB2013/001885 instead of making it in a single step, using a trifunctional reagent. This is not an optimal approach, but it was possible due to very high yield of each reaction. Polymer was first reacted with H-phosphonate of tritylated diethyleneglycol. It is important to use a starting glycol containing more than two carbons; otherwise, the free OH group in the final product, after removal of the trityl group, may destabilize the phosphotriester linkage. Oxidation of p 3 to p 5 was combined with the introduction of the azido group into the polymer, and it was done according to the described CCl 4 /pyridine/amine procedure, using 1-amino-6-azidohexane as a source of the azido group. The opposite order of incorporation of reactive groups using H phosphonate chemistry is also feasible. It is also recognized that H-phosphonate chemistry could be applied for the incorporation of any of existing reactive groups, if other factors of interest like hydrolytic stability of the starting reagents, or costs of syntheses, are deciding. Conjugates This document also provides conjugates that include a functionalized polymer as provided herein and a biologically active molecule. In some embodiments, a biologically active molecule is a TNF inhibitor or a derivative thereof. As used herein, the term "TNF inhibitor" includes antibodies or fusion proteins that bind to TNF alpha (e.g, human TNF alpha). Non-limiting examples of include etanercept (Enbrel@, Amgen and Pfizer); infliximab (Remicade@, Janssen Biotech, Inc.); adalimumab (Humira@, Abbott Laboratories); certolizumab pegol (Cimzia@); and Golimumab (Simponi@, Janssen Biotech, Inc.). In some embodiments, a biologically active moleculs is insulin or a derivative thereof. Derivatives of insulin include, for example, insulin analogs that contain one or more amino acid substitutions, and modifications to insulin or insulin analogs to include, for example, a moiety reactive with M or R on the polymer (as described above for compounds (1), (2), and (3)). Insulin and analogs can be recombinantly produced (e.g., in a non-pathogenic organism). Non limiting examples of suitable insulin analogs include insulin lispro (HUMALOG, sold by Eli Lilly), insulin aspart ("NovoLog/NovoRapid" sold by Novo Nordisk), insulin detemir (Levemir@ sold by Novo Nordisk), Insulin glargine (Lantus@, sold by Sanofi-Aventis), and insulin glulisine (Apidra@, sold by Sanofi-Aventis). 105 WO 2013/186632 PCT/IB2013/001885 Insulin lispro is a rapid-acting human insulin analog used to lower blood glucose levels, and contains a substitution of lysine for proline at position B28, and a substitution of proline for lysine at position B29. Insulin aspart is a rapid-acting human insulin analog used to lower blood glucose levels, and contains a substitution of an aspartic acid for proline at position B28. Insulin detemir is a long-acting insulin analog used for maintaining the basal level of insulin. Insulin determir contains myristic acid is bound to the lysine at position B29. Insulin glargine is a long-acting insulin analog used for lower blood glucose, and contains a substitution of a glycine for asparagine at position A21 and contains two arginines added to the C-terminus of the B-chain. Insulin glulisine is a rapid acting human insulin analog used to lower blood glucose levels, and contains a substitution of a lysine for asparagine at position B3 and the substitution of a glutamic acid for lysine at position B29. In some embodiments, a biologically active molecule is omalizumab or a derivative thereof. Omalizumab is sold under the trade name Xolair (Novartis Pharmaceuticals, East Hanover, NJ; Genentech Inc., South San Francisco, CA), and is a recombinant humanized monoclonal antibody having a molecular weight of approximately 150,000 Da. See Substance Identifier 47206967. See also U.S. Patent No. 6,267,958. "Antibody" as the term is used herein refers to a protein that generally comprises heavy chain polypeptides and light chain polypeptides. Antigen recognition and binding occurs within the variable regions of the heavy and light chains. Single domain antibodies having one heavy chain and one light chain and heavy chain antibodies devoid of light chains are also known. A given antibody comprises one of five types of heavy chains, called alpha, delta, epsilon, gamma and mu, the categorization of which is based on the amino acid sequence of the heavy chain constant region. These different types of heavy chains give rise to five classes of 15 antibodies, IgA (including IgAl and IgA2), IgD, IgE, IgG (IgGI, IgG2, IgG3 and IgG4) and IgM, respectively. A given antibody also comprises one of two types of light chains, called kappa or lambda, the categorization of which is based on the amino acid sequence of the light chain constant domains. Omalizumab is an IgGI antibody and contains kappa chains. IgG antibodies generally contain two identical heavy chains and two identical light chains and two antigen 106 WO 2013/186632 PCT/IB2013/001885 combining domains, each composed of a heavy chain variable region (VH) and a light chain variable region (VL). "Humanized antibody" refers to an antibody that has been engineered to comprise one or more human framework regions in the variable region together with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) of the heavy and/or light chain. In some embodiments, a humanized antibody comprises sequences that are entirely human except for the CDR regions. Humanized antibodies are typically less immunogenic to humans, relative to non-humanized antibodies, and thus offer therapeutic benefits in certain situations. For Omalizumab, the CDRs are derived from the parent murine monoclonal antibody and the constant regions of Omalizumab are derived from IgG 1 . Derivatives of Omalizumab include, for example, modifications to Omalizuabinsulin or insulin analogs to include, for example, a moiety reactive with M or R on the polymer (as described above for compounds (1), (2), and (3)) or a fragment of Omalizumab. A fragment of Omalizumab refers to a polypeptide derived from the heavy or light chain of Omalizumab that lacks all of part of at least one chain of Omalizumab. As the term as used herein encompasses fragments that comprise single polypeptide chains derived from antibody polypeptides (e.g., a heavy or light chain antibody polypeptide), it will be understood that an antibody fragment may not, on its own, bind an antigen. For example, an antibody fragment may comprise that portion of a heavy chain antibody polypeptide that would be contained in a Fab fragment; such an antibody fragment most commonly will not bind an antigen unless it associates with another antibody fragment derived from a light chain antibody polypeptide (e.g., that portion of a light chain antibody polypeptide that would be contained in a Fab fragment) and reconstitutes the antigen-binding site. Non-limiting examples of antibody fragments can include, for example, polypeptides that would be contained in Fab fragments, F(ab')2 fragments, or scFv (single chain Fv) fragments. In some embodiments, a biologically active molecule is a clotting factor or a derivative thereof. Derivatives of a clotting factor include, for example, a clotting factor analog that contains one or more amino acid substitutions relative to the corresponding human clotting factor, and modifications to the clotting factor or clotting factor analog to include, for example, a moiety reactive with M or R on the polymer (as described above for compounds (1), (2), and 107 WO 2013/186632 PCT/IB2013/001885 (3)). Clotting factors and analog thereof s can be recombinantly produced (e.g., in a non pathogenic organism). In some embodiments, a polypeptide that boosts red or white blood cell production. Examples of polypeptides that boost red blood cell production include epoetin alpha (Epogen@, Amgen), epoetin beta (NeoRecormon@, Roche), epoetin theta (Eporatio@), epoetin zeta and darbepoetin alpha (Aranesp@, Amgen)). Examples of polypeptides that boost white blood cell production include filgrastim (e.g., Neupogen@). Such polypeptides can be recombinantly produced. Derivatives of a polypeptide that boosts red or white blood cell production include, for example, an analog that contains one or more amino acid substitutions relative to the corresponding human polypeptide, and modifications to the polypeptide or analog to include, for example, a moiety reactive with M or R on the polymer (as described above for compounds (1), (2), and (3)). This document also provides conjugates that include a functionalized polymer as provided herein and a biologically active molecule such as an antibody or a derivative thereof. Non-limiting examples of antibodies that can be conjugated to a functionalized polymer include, abatacept (Orencia@), alemtuzumab (marketed as Campath, MabCampath or Campath-1H); basiliximab (Simulect@), belimumab (Benlysta@), besilesomab (Scintimun@), bevacizumab (Avastin@), canakinumab (Ilaris@), catumaxomab (Removab@), cetuximab (Erbitux@), denosumab (Prolia@, Xgeva@), eculizumab (Soliris@), ipilimumab (also known as MDX-010 or MDX-101, Yervoy@), natalizumab (Tysabri@), ofatumumab (Arzerra@), palivizumab (Synagis@), panitumumab (Vectibix@), ranibizumab (Lucentis@), rituximab (Rituxan@, MabThera@), tocilizumab (Actemra@, RoActemra@), trastuzumab (Herceptin@), and ustekinumab (Stelara@). Abatacept is sold under the trade name Orencia@ by Bristol~Myers Squibb Company, and is a fusion protein of the Fc region of IgGI fused to the extracellular domain of CTLA-4. It is a selective T cell costimulation modulator indicated for the treatment of adult rheumatoid arthritis and for juvenile idiopathic arthritis (JIA) in pediatric patients six years of age and older. Alemtuzumab is sold under the trade name Campath@, MabCampath@, or Campath-1H@ by Genzyme. Alemtuzumab is a recombinant DNA-derived humanized monoclonal antibody that is directed against the 21-28 kD cell surface glycoprotein,CD52. It is an IgGI kappa 108 WO 2013/186632 PCT/IB2013/001885 antibody with human variable framework and constant regions, and complementarity determining regions from a murine (rat) monoclonal antibody (Campath-IG). Campath is produced in mammalian cell (Chinese hamster ovary) suspension culture in a medium containing neomycin, and is used in the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma. It is also used in some conditioning regimens for bone marrow transplantation, kidney transplantation and islet cell transplantation. Basiliximab is sold under the trade name Simulect@ by Novartis Pharmaceuticals. It is a chimeric mouse-human monoclonal antibody to the a chain (CD25) of the IL-2 receptor of T cells. It is used to prevent rejection in organ transplantation, especially in kidney transplants. Belimumab is sold under the trade name Benlysta@ (Human Genome Sciences, Inc. and The GlaxoSmithKline Group of Companies). It is a monoclonal antibody that inhibits Beta cell activating factor (BAFF), and is used to treat adults with active systemic lupus erythematosus (SLE or lupus). Besilesomab is sold under the trade name Scintimun® (CiS Bio International). It is a mouse monoclonal antibody labeled with the radioactive isotope technetium-99m. It is used to detect inflammatory lesions and metastases. It binds to IgGi. Bevacizumab is sold under the trade name Avastin® by Genentech/Roche. It is a humanized monoclonal antibody that inhibits VEGF-A and slows angiogenesis. It is used to treat various cancers, including colorectal, lung, breast, glioblastoma, kidney, and ovarian cancer. Canakinumab is sold under the trade name Ilaris@ by Novartis. It is a human monoclonal antibody targeted at interleukin- 1 beta. It has no cross-reactivity with other members of the interleukin- 1 family, including interleukin- 1 alpha. Canakinumab is used to treat autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle Wells syndrome, and neonatal-onset multisystem inflammatory disease. Catumaxomab is sold under the trade name Removab® by TRION Pharma GmbH. It is a rat-mouse hybrid monoclonal antibody that is used to treat malignant ascites, which typically occurs in patients with metastasizing cancer. Catumaxomab has two binding specificities directed at epithelial cell adhesion molecule (EpCAM) and the T-cell antigen CD3. See Sebastian et al., Drugs Today (Barc). 45(8):589-97 (2009). 109 WO 2013/186632 PCT/IB2013/001885 Cetuximab is sold under the trade name Erbitux@ by Bristol-Myers Squibb and Eli Lilly and Company. It is a chimeric (mouse/human) monoclonal antibody that binds to the epidermal growth factor receptor (EGFR) and acts as an inhibitor. Cetuximab can be used for treatment of metastatic colorectal cancer and head and neck cancer. Denosumab is sold under the trade name Prolia@ and Xgeva@ by Amgen. It is a fully human monoclonal antibody that targets RANKL (RANK ligand), a protein that acts as the primary signal for bone removal, and is used for the treatment of osteoporosis, treatment-induced bone loss, bone metastases, rheumatoid arthritis, multiple myeloma, and giant cell tumor of bone. Eculizumab is sold under the trade name Soliris@ by Alexion Pharmaceuticals. It is a recombinant humanized monoclonal antibody that contains human constant regions from human IgG 2 sequences and human IgG 4 sequences and murine complementarity-determining regions grafted onto the human framework light- and heavy-chain variable regions. Eculizumab is composed of two 448 amino acid heavy chains and two 214 amino acid light chains and has a molecular weight of approximately 148 kDa. Eculizumab specifically binds the complement protein C5. Eculizumab has been shown to be effective in treating paroxysmal nocturnal hemoglobinuria (PNH), a rare and sometimes life threatening disease of the blood. Binding of Eculizumab to C5 inhibits its cleavage by the C5 convertase, and prevents the generation of the terminal complement complex C5b-9. It is this terminal complement complex that causes intravascular hemolysis in people with PNH. Ipilimumab (also known as MDX-010 or MDX-101) is sold under the trade name Yervoy@ by Bristol-Myers Squibb. It is a fully human monoclonal antibody that binds to CTLA-4 (cytotoxic T lymphocyte-associated antigen 4), and is used to treat melanoma (e.g., metastatic melanoma or unresectable melanoma). Natalizumab is sold under the trade name Tysabri@ by Biogen Idec and tlan. It is a humanized monoclonal antibody against the cell adhesion molecule a4-integrin. Natalizumab is used in the treatment of multiple sclerosis and Crohn's disease. Ofatumumab is sold under the trade name Arzerra@ by Genmab. It is a human monoclonal antibody that specifically binds the CD20 protein and inhibits early-stage B lymphocyte activation. Ofatumumab is used for treating chronic lymphocytic leukemia, 110 WO 2013/186632 PCT/IB2013/001885 follicular non-Hodgkin's lymphoma, diffuse large B cell lymphoma, rheumatoid arthritis, and relapsing remitting multiple sclerosis. Palivizumab is sold under the trade name Synagis@ by MedImmune. It is a humanized monoclonal antibody (IgG1 kappa) directed against an epitope in the A antigenic site of the F protein of RSV. Palivizumab is a composite of human (95%) and murine (5%) antibody sequences. Panitumumab is sold under the trade name Vectibix@ by Amgen. It is a fully human monoclonal antibody specific to the epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1 and HERI in humans), and is used to treat metastatic colorectal cancer. Ranibizumab is sold under the trade name Lucentis® by Novartis. It is a monoclonal antibody fragment (Fab) derived from the same parent mouse antibody as bevacizumab and targets VEGF-A. Ranibizumab is an anti-angiogenic and can be used to treat age-related macular degeneration (e.g., AMD and ARMD). Rituximab is sold under the trade names Rituxan® and MabThera® by Biogen Idec and Genentech. It is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of B cells. Rituximab can be used to treat lymphomas, leukemias, transplant rejection and some autoimmune disorders. Tocilizumab is sold under the trade names Actemra® and RoActemra@ by Hoffmann-La Roche and Chugai. It is a humanized monoclonal antibody that binds the interleukin-6 receptor (IL-6R) and is used as an immunosuppressive to treat rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis. Trastuzumab is sold under the trade name Herceptin® by Genentech. It is a humanized monoclonal antibody that binds domain IV of the extracellular segment of the HER2/neu receptor. It typically is used to treat breast cancer. Ustekinumab is sold under the trade name Stelara® by Centocor. It is human monoclonal antibody that is directed against interleukin 12 and interleukin 23, and is used to treat psoriasis. In certain embodiments, "Chimeric antibody" as the term is used herein refers to an antibody that has been engineered to comprise a human constant region. Chimeric antibodies are typically less immunogenic to humans, relative to non-chimeric antibodies, and thus offer therapeutic benefits in certain situations. 111 WO 2013/186632 PCT/IB2013/001885 Derivatives of an antibody include, for example, modifications to the antibody to include, for example, a moiety reactive with M or R on the polymer (as described above for compounds (1), (2), and (3)) or a fragment of the antibody. A fragment of the antibody refers to a polypeptide derived from the heavy or light chain of the antibody that lacks all of part of at least one chain of the antibody. As the term as used herein encompasses fragments that comprise single polypeptide chains derived from antibody polypeptides (e.g., a heavy or light chain antibody polypeptide), it will be understood that an antibody fragment may not, on its own, bind an antigen. For example, an antibody fragment may comprise that portion of a heavy chain antibody polypeptide that would be contained in a Fab fragment; such an antibody fragment most commonly will not bind an antigen unless it associates with another antibody fragment derived from a light chain antibody polypeptide (e.g., that portion of a light chain antibody polypeptide that would be contained in a Fab fragment) and reconstitutes the antigen-binding site. Non limiting examples of antibody fragments can include, for example, polypeptides that would be contained in Fab fragments, F(ab')2 fragments, or scFv (single chain Fv) fragments. This document also provides conjugates that include a functionalized polymer as provided herein and a biologically active molecule such as therapeutic agents, small molecules such as synthetic drugs, oligo- and polypeptides, oligonucleotides, coding DNA sequences, antisense DNA sequences, mRNAs, antisense RNA sequences, RNAIs, and siRNAs, carbohydrates, lipids, growth factors, enzymes, transcription factors, toxins, antigenic peptides (as for vaccines), antibodies, and antibody fragments. For example, a biologically active molecule can be agalsidase alfa (Replagal@ ), agalsidase beta (Fabrazyme@), alglucosidase alfa (Myozyme@), anakinra (Kineret@), bortezomib (Velcade@), conestat alfa (Ruconest@), choriogonadotropin alfa (e.g., Ovidrel@), cinacalcet (Sensipar@), Corifollitropin alfa (Elonva@), dibotermin alfa (InductOs@), eltrombopag (Promacta@), eptotermin alfa (Osigraft@), erlotinib (Tarceva@), follitropin alfa /lutropin alfa, follitropin beta, galsulfase (Naglazyme@), Gefitinib (Iressa@), Human normal immunoglobulin, human normal immunoglobulin (ivig), human normal immunoglobulin (SCIg), idursulfase (Elaprase@), Imiglucerase (Cerezyme@), interferon alfa-2b, interferon beta-Ia, interferon beta- Ib, Lapatinib (Tykerb@, Tyverb@), Laronidase (Aldurazyme@), Pazopanib (Votrient), pegaptanib, pegfilgrastim, peginterferon alfa-2b, 112 WO 2013/186632 PCT/IB2013/001885 Rasburicase (Elitek@), Reteplase (Retavase@), Somatropin, Sorafenib (Nexavar@), Tenecteplase (TNKase), thyrotropin alfa, Vandetanib (Caprelsa@), and Vemurafenib (Zelboraf@). Agalsidase alfa and Agalsidase beta (Fabrazyme@) are recombinant human a galactosidase A enzymes, and can be used for long-term enzyme replacement therapy in patients with Fabry's disease, which is caused by the lack of alpha-galactosidase A. Aglucosidase alfa is recombinant alpha glucoside, and can be used for enzyme replacement therapy in patients with Pompe's disease. Anakinra (Kineret @) is an interleukin 1 receptor antagonist, and can be used to reduce the pain and swelling associated with rheumatoid arthritis. Bortezomib (Velcade @) is an N-protected dipeptide used to treat people with multiple myeloma Conestat alfa is a recombinant human C1 obtained from the milk of transgenic rabbits. It can be used to treat hereditary angioneurotic edema. Choriogonadotropin alfa (Ovidrel@) is human chorionic gonadotropin (HCG), and can be used to help produce mature eggs during in vitro fertilization and stimulate ovulation (release of an egg). Cinacalcet is a calcimimetic agent that increases the sensitivity of the calcium-sensing receptor to activation by extracellular calciumcinacalcet. See e.g., US Patent No. 6,211,244. It can be used to treat secondary hyperparathyroidism. Corifollitropin alfa (Elonva@) is a modified follicle stimulating hormone (FSH), and can be used to stimulate egg production during infertility treatments. Dibotermin alfa (InductOs@) is recombinant human bone morphogenetic protein 2 and can be used to promote new bone formation and for the treatment of bony defects. Eltrombopag (Promacta@) is a small molecule agonist of the c-mpl (TpoR) receptor, and is used to increase the number of platelets (cells that help the blood clot) in order to decrease the risk of bleeding in people who have chronic idiopathic thrombocytopenic purpura. Eptotermin alfa (Osigraft@) is recombinant human bone morphogenetic protein 7 (hrBMP-7) (also referred to as Osteogenic protein-I (OP-1)) and is used to aid healing of bone. 113 WO 2013/186632 PCT/IB2013/001885 Erlotinib (Tarceva@) is a reversible tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR). It is used for treatment of nonsmall cell lung cancer and pancreatic cancer (in combination with gemcitabine). Follitropin alfa /lutropin alfa contains a recombinant follicle stimulating hormone (FSH) and recombinant luteinizing hormone (LH). Follitropin beta is another recombinant FSH. Galsulfase is a recombinant form of human N-acetylgalactosamine 4-sulfatase (Naglazyme@; BioMarin) and is used for treatment of mucopolysaccharidosis type VI Gefitinib (Iressa@) is an EGFR inhibitor that is used for treatment of cancer (e.g., nonsmall cell lung cancer). Idursulfase (Elaprase@) is the recombinant lysosomal enzyme iduronate-2-sulfatase, and is used to treat Hunter syndrome (Mucopolysaccharoidosis II)). Imiglucerase (Cerezyme@) is a recombinant analog of human P-glucocerebrosidase, and is used to treat Gaucher's disease. Lapatinib (Tykerb@, Tyverb@) is a dual tyrosine kinase inhibitor that interrupts the HER2 growth receptor pathway, and is used to treat breast cancer. Laronidase (Aldurazyme@) is a recombinant polymorphic variant of the human enzyme a-L-iduronidase enzyme, and is used to treat mucopolysaccharoidosis. Pazopanib (Votrient) is a potent and selective multi-targeted receptor tyrosine kinase inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-a/p, and c-kit that blocks tumor growth and inhibits angiogenesis. Rasburicase (Elitek@) is a recombinant version of urate oxidase, an enzyme which occurs in many mammals but not in humans. It can be used to treat hyperuricemia in patients after cancer treatment. Reteplase (Retavase@) is a recombinant non-glycosylated form of human tissue plasminogen activator, which has been modified to contain 357 of the 527 amino acids of the original protein. It is a thrombolytic drug and used to treat heart attacks. Rilonacept (Arcalyst@) is a dimeric fusion protein consisting of the extracellular domain of human interleukin-1 receptor and the FC domain of human IgGI that binds and neutralizes IL 1. It can be used to treat gout. 114 WO 2013/186632 PCT/IB2013/001885 Sorafenib (Nexavar@) is a small molecular inhibitor of several tyrosine protein kinases (VEGFR and PDGFR) and Raf kinases. It can be used to treat renal cell carcinoma and liver cancer. Tenecteplase (TNKase) is a recombinant tissue plasminogen activator analog, and is used as a thrombolytic drug. Vandetanib (Caprelsa@) is an an antagonist of the vascular endothelial growth factor receptor (VEGFR) and the epidermal growth factor receptor (EGFR), and can be used to treatment of thyroid cancer. Vemurafenib (Zelboraf@) is a B-Raf enzyme inhibitor and used for treatment of melanoma. Numerous other examples of biologically active molecules will be apparent to the skilled artisan. Derivatives of a biologicaly active molecule include modifications to the biologically active molecule to include, for example, a moiety reactive with M or R on the polymer (as described above for compounds (1), (2), and (3)) Preparation of conjugates between a functionalized polymer provided herein and a biologically active molecule or a derivative thereof occurs through a coupling reaction between the reactive group on the polymer with a biologically active molecule or derivative thereof. One of skill in the art would appreciate that there are many ways to couple a biologically active molecule or a derivative thereof with the functional polymers described herein. For example, a biologically active molecule can be modified to introduce thiols at the places of reactive amino groups (e.g., at lysine residues) that can subsequently react with a maleimido or iodoacetamido group activated functionalized polymer described herein. A biologically active molecule also can be derivatized to incorporate hydrazo functions that can subsequently react with an aldehyde/keto functionalized polymer described herein. In another method, a biologically active molecule is reacted with a reagent that introduces an azido function at amino groups. The resulting azido modified biologically active molecule (e.g., antibody, polypeptide) then can be further reacted with an alkyne-derivatized functionalized polymer to obtain a conjugate via the Click-reaction. 115 WO 2013/186632 PCT/IB2013/001885 In another method, a biologically active molecule can be reacted with a reagent introducing a diene/dienophile that can subsequently react with a diene/dienophile activated functionalized polymer described herein to produce a conjugate after the Diels-Alder reaction. In another method, the functionalized polymer is directly linked to a biologically active molecule or a derivative thereof. For example, a functional polymer (e.g., PEG) containing a carboxyl group that is previously activated by a reactive ester group like N-hydroxysuccinimide (NHS) can be conjugated to a biologically active molecule or a derivative thereof. Generally, a functionalized polymer described herein can include an activated ester as M R, and can react randomly with protein amino groups (e.g., lysines) to form covalent linkages. Many activating groups are available commercially as a wide variety of leaving groups are known. Non-limiting examples of leaving groups include p-nitrophenol and NHS. Attaching of PEGs to antibodies is often made randomly because the IgG molecule often demands several polymers to be efficiently protected. In another method, aldehyde-containing functionalized polmers can form a Schiffs base with amino groups on the protein. The Schiffs base is further selectively reduced with sodium cyanoborohydride in a well known reaction. In another method, periodate can be used to oxidize carbohydrates on a biologically active molecule to aldehydes, followed by addition of hydrazo derivatized PEG to be attached. The hydrazo group reacts with the aldehydes to produce a stable hydrazide link. In one embodiment, a biologically active molecule conjugate, or a pharmaceutically acceptable salt thereof includes a water-soluble, non-peptidic, and non-nucleotidic polymer backbone as in a structure of formula (9): E R1 A-0-P-Zi-L-L 2 -B Z2 K G or a salt thereof, wherein: 116 WO 2013/186632 PCT/IB2013/001885 A is the point of covalent bonding to one terminus of the polymer backbone; E is 0 or S; K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic separation handle; Z' and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; R 1 is absent or a hydrophobic separation handle, wherein only one of R 1 and G can be a hydrophobic separation handle; L2 is a covalent linking moiety between L on the polymer backbone and B (e.g., amide, carbamide, ester, oxime, thioether, dithioether, secondary amine, 1,2, 4-triazol, or hydrazide linking moiety); and B is a biologically active molecule or a derivative thereof. Such a conjugate can be prepared by reacting a biologically active molecule or a derivative thereof with a preparation comprising a water-soluble, non-peptidic, and non nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (1) (as described above) under conditions suitable for group M to react with a biologically active molecule or the derivative thereof. The group M can be hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide. In some embodiments, the group reactive with a biologically active molecule or derivative thereof is carboxyl and R is absent or is N-hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl. In one embodiment, a conjugate, or a pharmaceutically acceptable salt thereof, has a structure of formula (10): El E
B
1
-L
3
-L
1
-Z
3 -P- polymer-O-P-Z-L-L 2 -B 1 1 z4 2 Gi-K1 K--G (10) or a salt form thereof, 117 WO 2013/186632 PCT/IB2013/001885 wherein polymer has a linear, water-soluble, non-peptidic, and non-nucleotidic polymer backbone (e.g., PEG), wherein each linking group is bonded at a different terminus of the polymer; E and E' are independently 0 or S; K and K 1 are independently selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G and G 1 are independently absent or are selected from the group consisting of: alkoxy and a hydrophobic separation handle; each pair of Z' and Z 2 and Z 3 and Z 4 are independently selected from 0 and NH, wherein only one of each pair of Z' and Z 2 and Z 3 and Z 4 can be NH; L and L' are independently selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; L2 is a covalent linking moiety (e.g., amide, carbamide, ester, oxime, thioether, dithioether, secondary amine, 1,2, 4-triazol, or hydrazide linking moiety); between L on the polymer backbone and B; L 3 is a covalent linking moiety (e.g., amide, carbamide, ester, oxime, thioether, dithioether, secondary amine, 1,2, 4-triazol, or hydrazide linking moiety) between L on the polymer backbone and B'; and B and B are independently a biologically active molecule or a derivative of a biologically active molecule (e.g., a TNF inhibitor, a derivative of a TNF inhibitor, insulin, a derivative of insulin a clotting factor, a derivative of a clotting factor, omalizumab, a derivative of malizumab, a polypeptide that boosts red or white cell production, or a derivative of a polypeptide that boosts red or white cell product, an antibody, or a derivative of an antibody), a biologic other than the biologically active molecule (e.g., an antibody or a polypeptide other than the biologically active molecule), a drug, a detectable group, or a separation moiety, wherein at least one of B and B 1 is a biologically active molecule or a derivative of a biologically active molecule. For example, B can be selected from the group consisting of a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, or an antibody; and B 1 can be a derivative of a biologically active molecule selected from the group group consisting of a TNF inhibitor, insulin, omalizumab, a clotting factor, a polypeptide that boosts red or white cell production, and an antibody; B and B 1 can both be a biologically active molecule; one of B and B 1 is a biologically active molecule and the other is a separation moiety; one of B and B 1 is a biologically active molecule and the other is a different biologic; one of B and B 1 is a biologically active molecule and the other is a detectable group; or one of B and B 1 is a biologically active molecule and the other is a drug. 118 WO 2013/186632 PCT/IB2013/001885 Such a conjugate can be prepared by reacting a biologically active molecule or a derivative thereof with a preparation comprising a compound of formula (2) (as described above) under conditions suitable for group M to react with a biologically active molecule or the derivative thereof. The group M can be hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide. In some embodiments, the group reactive with a biologically active molecule or derivative thereof is carboxyl and R is absent or is N hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl. In one embodiment, a conjugate, or a pharmaceutically acceptable salt thereof, has a structure of formula (11): E
B
1 - L 4 polymer O-P Z- L-L Z2 B K- G (11) or a salt form thereof, wherein polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer backbone (e.g., a PEG polymer), wherein M2 and the phosphonate-derived functional group are bonded at a different terminus of said polymer; E and El are independently 0 or S; K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene; G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic separation handle; ZI and Z 2 are independently selected from 0 and NH, wherein only one of Z' and Z 2 can be NH; L is selected from the group consisting of: a divalent radical of nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene; L2 is a covalent linking moiety (e.g., amide, carbamide, ester, oxime, thioether, dithioether, secondary amine, 1,2, 4-triazol, or hydrazide linking moiety); between L on the polymer backbone and B; L 4 is a covalent linking moiety (e.g., amide, carbamide, ester, oxime, thioether, dithioether, secondary amine, 1,2, 4-triazol, or hydrazide linking moiety); between L on the polymer backbone and B 1 ; and B and B are independently a biologically active molecule or a derivative of a biologically active molecule (e.g., a TNF inhibitor, a derivative of a TNF 119 WO 2013/186632 PCT/IB2013/001885 inhibitor, insulin, a derivative of insulin, omalizumab, a derivative of omalizumab, a clotting factor, a derivative of a clotting factor, a polypeptide that boosts red or white cell production, a derivative of a polypeptide that boosts red or white cell production, an antibody, or a derivative of an antibody), a biologic other than a biologically active molecule, a drug, a detectable group, a separation moiety, wherein at least one of B and B 1 is a biologically active molecule or a derivative of a biologically active molecule. For example, B can be a biologically active molecule and B 1 can be a derivative of a biologically active molecule; B and B 1 can both be a biologically active molecule; one of B and B 1 is a biologically active molecule and the other is a separation moiety; one of B and B 1 is a biologically active molecule and the other is a different biologic; one of B and B 1 is a biologically active molecule and the other is a detectable group; or one of B and B 1 is a biologically active molecule and the other is a drug. Preparations Also provided herein are preparations that include a compound or conjugate provided herein. In some embodiments, a preparation can include at least 50% of a compound or conjugate by weight. For example, a preparation can include at least 60%, at least 65%, at least 7 0
%
, at least 75% at least 77%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% by weight of the compound or conjugate. In some embodiments, the compound or conjugate is essentially pure in the preparation. A preparation can be a solution, a reaction mixture, a chromatographic eluent, a solid (e.g., a power or crystalline form of the preparation), or any other mixture that includes a compound or conjugate in the appropriate amount or level or purity. Pharmaceutically Acceptable Salts and Compositions This document also provides pharmaceutically acceptable salts of the compounds and conjugates provided herein. Examples of pharmaceutically acceptable salts of a compound or a conjugate provided herein include acid addition salts and base salts of the same. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, without limitation, the acetate, adipate, aspartate, benzoate, besylate, 120 WO 2013/186632 PCT/IB2013/001885 bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, hydrogen phosphate, isethionate, D- and L-lactate, malate, maleate, malonate, mesylate, methylsulphate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen, phosphate/phosphate dihydrogen, pyroglutamate, saccharate, stearate, succinate, tannate, D- and L-tartrate, 1-hydroxy-2-naphthoate tosylate, and xinafoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include, without limitation, the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. A conjugate, as provided herein, can be formulated into a pharmaceutical composition that includes an effective amount of a conjugate and a pharmaceutically acceptable excipient. Also provided herein are pharmaceutical compositions that include an effective amount of a compound as provided herein, wherein M is a detectable functional group, and a pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutical excipients suitable for administration of the conjugates and compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block 121 WO 2013/186632 PCT/IB2013/001885 polymers, and wool fat. Cyclodextrins such as a-, P, and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-b cyclodextrins, or other solubilized derivatives can also be advantageously used to enhance delivery of a compound or conjugate provided herein. In some embodiments, the excipient is a physiologically acceptable saline solution. A pharmaceutical composition can be, in one embodiment, formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal ointments, creams, gels, and patch preparations and dry powder inhalers (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126). The concentration of a compound or conjugate in a pharmaceutical composition will depend on absorption, inactivation, and excretion rates of the compound or conjugate, the physicochemical characteristics of the compound or conjugate, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. The pharmaceutical composition may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular patient, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or conjugates. The pharmaceutically therapeutically active compounds or conjugates are, in one embodiment, formulated and administered in unit-dosage 122 WO 2013/186632 PCT/IB2013/001885 forms or multiple-dosage forms. Unit-dose forms as used herein refer to physically discrete units suitable for human and animal patients and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound or conjugate sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing a compound or conjugate as provided herein and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. If desired, a pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Dosage forms or compositions containing a compound or conjugate provided herein in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.00 1 %-100% active ingredient, in one embodiment 0.1-95%, in another embodiment 75-85%. Pharmaceutical compositions suitable for the delivery of a compound or conjugate provided herein and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). Methods of Treatment 123 WO 2013/186632 PCT/IB2013/001885 Conjugates of a biologically active molecule or a derivative thereof and a functionalized polymer as provided herein, and combinations thereof (e.g., two or more conjugates of a biologically active molecule or a derivative thereof) can be used to treat a patient as appropriate for the particular biologically active molecule (e.g., a patient having a disorder selected from the group consisting of an inflammatory disease; diabetes; asthma; hemophilia (e.g., hemophilia A or hemophilia B), a deficiency in a clotting factor (e.g., factor VII deficiency) or other disorder affecting coagulation or clotting; with anemia, e.g., a human patient having anemia resulting from chemotherapy or kidney disease). In some embodiments, conjugates of TNF inhibitors or a derivative thereof and a functionalized polymer as provided herein, and combinations thereof (e.g., two or more conjugates of a TNF inhibitor or a derivative thereof) can be used to treat a patient having an inflammatory disease, e.g., rheumatoid arthritis, psoriasis such as plaque psoriasis or chronic psoriasis, psoriatic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, Crohn's disease, or ulcerative colitis. As used herein, treating inflammatory disease refers to reducing the severity of the disease or slowing progression of the disease. The methods described herein include administering to the patient an effective amount of the conjugate. An effective amount of conjugate(s) or a pharmaceutical formulation containing a conjugate can be any amount that reduces the severity of the disease or slows progression of the disease while not inducing significant toxicity in the patient. Effective amounts of conjugates or a pharmaceutical composition can be determined by a physician, taking into account various factors that can modify the action of drugs such as overall health status, body weight, sex, diet, time and route of administration, other medications, and any other relevant clinical factors. In some embodiments, conjugates of insulin or a derivative thereof and a functionalized polymer as provided herein, and combinations thereof (e.g., two or more conjugates of insulin or a derivative thereof) can be used to treat a patient having diabetes, e.g., a human patient having type 1 or type 2 diabetes. As used herein, treating diabetes refers to helping to control blood glucose levels. The methods described herein include administering to the patient an effective amount of the conjugate or combinations of conjugates. An effective amount of conjugate(s) or a pharmaceutical formulation containing a conjugate can be any amount that helps reduce or maintain blood glucose levels while not inducing significant toxicity in the patient. Effective 124 WO 2013/186632 PCT/IB2013/001885 amounts of conjugates or a pharmaceutical composition can be determined by a physician, taking into account various factors that can modify the action of drugs such as overall health status, body weight, sex, diet, time and route of administration, other medications, and any other relevant clinical factors. A conjugate or a pharmaceutical formulation containing a conjugate can be administered by any route, including, without limitation, oral or parenteral routes of administration such as intravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal, intraarterial, nasal, transdermal (e.g., as a patch), or pulmonary absorption. A conjugate described herein can be formulated as, for example, a solution, suspension, or emulsion with one or more pharmaceutically acceptable excipients suitable for the particular route of administration. Subcutaneous administration is particularly useful. In some embodiments, conjugates of omalizumab or a derivative thereof and a functionalized polymer as provided herein, and combinations thereof (e.g., two or more conjugates of omalizumab or a derivative thereof) can be used to treat a patient having asthma. As used herein, treating asthma refers to reducing the severity of the disease (e.g., reducing the incidence of asthma exacerbations). Treatment with Omalizumab limits the release of mediators of the allergic response and reduces the number of FcRI receptors on basophils in atopic patients. Treatment with Omalizumab also can increase the expiratory volume in the first second of a forced expiration (FEV 1 ), which is an indicator of the ability to exhale rapidly and completely. The methods described herein include administering to the patient an effective amount of the conjugate. An effective amount of conjugate(s) or a pharmaceutical formulation containing a conjugate can be any amount that reduces the severity of asthma while not inducing significant toxicity in the patient. In some embodiments, conjugates of a clotting factor or a derivative thereof and a functionalized polymer as provided herein can be used to treat a patient (e.g., a human patient) diagnosed with hemophilia (e.g., hemophilia A or hemophilia B), a deficiency in a clotting factor (e.g., factor VII deficiency) or other disorder affecting coagulation or clotting. As used herein, treating refers to reducing the severity of the disease or disorder. 125 WO 2013/186632 PCT/IB2013/001885 Eptacog alfa (activated) (sold by Novo Nordisk) can be used to replace missing factor VII in patients with factor VII deficiency and also can be used in hemophilia patients who have developed inhibitors to factor VIII or IX. Factor VIII deficiency results in hemophilia A and Factor IX deficiency causes hemophilia B. Moroctocog alfa (glycoprotein (trade name ReFacto@, Xyntha@) are commercially available for Factor VIII replacement therapy. Factor VIII replacement therapy is limited due to development of high-titer inhibitory factor VIII antibodies in some patients. Nonacog alfa (Benefix@) is a human recombinant factor IX product used in treating hemophilia B patients. Antithrombin is a small glycoprotein that inactivates several enzymes of coagulation system. Its activity enhanced by heparin. Antithrombin alfa (e.g., ATryn@) is commercially available. In some embodiments, conjugates of a polypeptide that boosts red or white blood cell production or a derivative thereof and a functionalized polymer as provided herein can be used to treat a patient with anemia, e.g., a human patient having anemia resulting from chemotherapy or kidneythe group consisting of an inflammatory disease. Conjugates of a polypeptide that boosts white blood cell production or a derivative thereof and a functionalized polymer as provided herein, and combinations thereof (e.g., two or more conjugates of a polypeptide that boosts red or white blood cell production or a derivative thereof) can be used to treat a patient with neutropenia (e.g., neutropenia resulting from chemotherapy or bone marrow transplantation) or to increase the number of hematopoietic stem cells in the blood before collection. As used herein, treating anemia or neutropenia refers to reducing the severity of the disease. The methods described herein include administering to the patient an effective amount of the conjugate. An effective amount of a conjugate or a pharmaceutical formulation containing a conjugate can be any amount that reduces the severity of the disease or disorder while not inducing significant toxicity in the patient. Conjugates of an antibody or a derivative thereof and a functionalized polymer as provided herein, and combinations thereof (e.g., two or more conjugates of an antibody or a derivative thereof) can be used to treat a patient, e.g., a human patient. For example, a conjugate that includes Abatacept can be used for the treatment of arthritis (e.g., adult rheumatoid arthritis 126 WO 2013/186632 PCT/IB2013/001885 or JIA). A conjugate that includes Alemtuzumab can be used for treatment of CLL, CTCL, and T-cell lymphoma, or in a conditioning regimen for bone marrow transplantation, kidney transplantation, or islet cell transplantation. A conjugate that includes Basiliximab can be used to prevent rejection in organ transplantation, (e.g., during kidney transplants). A conjugate that includes Belimumab can be used to treat adults with active SLE. A conjugate that includes Besilesomab can be used to detect inflammatory lesions and metastases. A conjugate that includes Bevacizumab can be used to treat various cancers, including colorectal, lung, breast, glioblastoma, kidney, and ovarian cancer. A conjugate that includes Canakinumab can be used to treat autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle Wells syndrome, and neonatal-onset multisystem inflammatory disease. A conjugate that includes Catumaxomab can be used to treat malignant ascites. A conjugate that includes Cetuximab can be used for treatment of metastatic colorectal cancer and head and neck cancer. A conjugate that includes Denosumab can be used for the treatment of osteoporosis, treatment induced bone loss, bone metastases, rheumatoid arthritis, multiple myeloma, and giant cell tumor of bone. A conjugate that includes Eculizumab can be used to treat paroxysmal nocturnal hemoglobinuria (PNH). A conjugate that includes Ipilimumab can be used to treat melanoma (e.g., metastatic melanoma or unresectable melanoma). A conjugate that includes Natalizumab can be used in the treatment of multiple sclerosis and Crohn's disease. A conjugate that includes Ofatumumab can be used for treating chronic lymphocytic leukemia, follicular non-Hodgkin's lymphoma, diffuse large B cell lymphoma, rheumatoid arthritis, and relapsing remitting multiple sclerosis. A conjugate that includes Panitumumab can be used to treat metastatic colorectal cancer. A conjugate that includes Ranibizumab can be used to treat age-related macular degeneration (e.g., AMD and ARMD). A conjugate that includes Rituximab can be used to treat lymphomas, leukemias, transplant rejection and some autoimmune disorders. A conjugate that includes Tocilizumab can be used to treat rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis. A conjugate that includes Trastuzumab can be used to treat breast cancer. A conjugate that includes Ustekinumab can be used to treat psoriasis. As used herein, treating a particular disease or disorder refers to reducing the severity of the disease or slowing progression of the disease. 127 WO 2013/186632 PCT/IB2013/001885 Methods described herein can include monitoring the patient to, for example, determine if the severity or progression of the disease is improving with treatment. Synthesis Compounds and conjugates provided herein, including salts thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes. In some embodiments, a compound or conjugate is prepared using a method as provided herein. The reactions for preparing compounds and conjugates provided herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by the skilled artisan. Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Protecting Group Chemistry, 1 st Ed., Oxford University Press, 2000; March's Advanced Organic chemistry: Reactions, Mechanisms, and Structure, 5 Ih Ed., Wiley-Interscience Publication, 2001; and Peturssion, S. et al., "Protecting Groups in Carbohydrate Chemistry," J. Chem. Educ., 74(11), 1297 (1997) (each of which is incorporated herein by reference in their entirety. Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS) or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of 128 WO 2013/186632 PCT/IB2013/001885 methods, including high performance liquid chromatography (HPLC) ("Preparative LC-MS Purification: Improved Compound Specific Method Optimization" K.F. Blom, et al., J. Combi. Chem. 6(6) (2004), which is incorporated herein by reference in its entirety) and normal phase silica chromatography. EXAMPLES Example 1. Synthesis of isopropyl phosphorodichloridite was performed according to the modified literature procedure of Zwierzak and Koziara, Tetrahedron (1967), 23, 2243-2252. Dry isopropanol (2 mole) in 100 mL of dry diethyl ether was added dropwise to the vigorously stirred mixture of phosphorus trichloride (4 mole) in 200 mL of diethyl ether at 20 0 C. The mixture was allowed to warm up to room temperature, and was stirred at this temperature for 4 hours. The mixture was fractionally distilled at normal pressure to obtain the title product by collecting a colorless fraction having bp. 120-125 0 C. Example 2. Synthesis of isopropyl, N,N-diisopropylphosphoramidochloridite. Isopropyl phosphorodichloridite (56.5 g, 0.5 mole) in dry diethyl ether (300 mL) was placed in a IL round bottom flask and cooled to -30 C. To this vigorously stirred solution, dry diisopropyl amine (109 g, 1 mole) in diethyl ether (200 mL) was added dropwise (2 hr) at the above low temperature. The mixture was then allowed to warm to room temperature and was left at this temperature overnight. The thick white cake of diisopropylammonium hydrochloride was filtered off on a large filter funnel and washed with two portions of ether. The combined ether phase was evaporated and the residue was distilled at lowered pressure collecting a fraction boiling at 83-85 0 C (15 Torr). Example 3. The following is a general procedure used to prepare different phosphoramidite reagents. An appropriately protected alcohol (1 eq.) was placed in a round bottom flask and dried by coevaporation with toluene. The residue was dissolved in dry dichloromethane (DCM) (5 129 WO 2013/186632 PCT/IB2013/001885 mL/mmole), and dry triethylamine (TEA) (4 eq.) was added in one portion. To this solution, stirred at room temperature, isopropyl, N,N-diisopropylphosphoramidochloridite (1.5 eq.) in DCM (2 mL/mmole) was added dropwise. The mixture was stirred an additional 30 minutes and when TLC (DCM:Methanol:TEA 95:4:1) showed a complete consumption of the starting alcohol, methanol (5 eq.) was added in one portion and the mixture was stirred further for 30 minutes. The reaction mixture was partitioned between dichloromethane and an aqueous solution of sodium bicarbonate. The organic extracts were combined, evaporated, dried by coevaporation with toluene, and purified by flash column chromatography on silica gel using a gradient of ethyl acetate in hexane with addition of 2% TEA as an eluent. The appropriate fractions were collected, and evaporated. The residual solvents were removed under high vacuum, yielding products in the form of thick oils. The following starting alcohols were prepared according to the published literature and converted to the respective isopropyl phosphoramidites using the procedure outlined above: 1) An amidite useful for introduction of an amino group was prepared by modification of the method of Gaur, Nucleosides, Nucleotides & Nucleic Acids (1991), 10, (4), 895 - 909: MMTrs'N' 'OH 2 MMTr-N' 'O-P,N(iPr) 2 H H O-Pr 2) An amidite useful for introduction of an aminoxy group was prepared in a process similar to the synthesis of described above for aminating an amidite. The starting aminoxy alcohol was prepared from 6-bromhexanol according to Khomutov, Zhurnal Obshch Khimyi 1961, 31, 1992-1995: MMTr, N'OOH : MMTr,' N'O,--s-s , N(iPr)2 H H O-iPr 3) An amidite useful for introduction of a hydrazo group was prepared using a modified method of Raddetz et al., Nucleic Acids Res. 30, (21), 4793-4802: H 0 H 0 N(iPr) 2 MMTr N N 'OH - MMTr N ,'N O-Pr H H 130 WO 2013/186632 PCT/IB2013/001885 4) An amidite useful for introduction of a thiol was obtained following the procedure described by Connolly and Rider in Nucleic Acids Res. 1985, 13, (12), 4485-4502: TN(iPr) 2 r O-Pr 5) An amidite useful for introduction of a carboxyl group was prepared utilizing a chlorotrityl group for protection of a carboxyl group as was described by Kachalova et al. Helv. Chim. Acta 2002, 85, 2409-2416: 0 0 CITr-,. CITr-, ' Cr' , N(iPr)2 OH 0 PNOiPr 0-OiPr 6) An amidite useful for introduction of biotin was prepared following the methodology described by Krempsky et al., Tet. Lett., (1996), 37,(12), 4313-4316: 0 0 DMTr-N NH DMTr-N NH NO '^'H NN(iPr) 2 S N----"OH S S OP H H O-iPr 7) The following amidite, prepared in a method analogous to one described by Singh et al. J. Org. Chem. 2004, 69, 8544-8546, was used for incorporation of an aliphatic aldehyde group. The aldehyde (as an amide of glyoxalic acid) was obtained after oxidative cleavage of the cis-amine alcohol group: FMCH 0 H 0 ,N(iPr) 2 FMOC- N '^'OH FMOC-N N ^O' DMH- H O-Pr DMTr-O DMTr-O H 8) The following amidite can be used for incorporation of an aliphatic aldehyde group. This reagent was made in analogy to Spinelli et al. Nucleosides, Nucleotides and Nucleic Acids 2007, 26, 883-887. The aldehyde was obtained after acidic hydrolysis of the acetal bond and oxidation of the cis diol system: 131 WO 2013/186632 PCT/IB2013/001885
CH
3 -O
CH
3 -0 OH O__ 0 , N(iPr) 2 0-iPr 9) The following amidite, prepared by modification of method of Podyminogin et al., Nucl. Acids Res. 2001, 29, (24), 5090-5098, can be used for incorporation of an aromatic aldehyde group: O 3 ,^ OH 3 -O _ N(iPr) 2 0H 3 -O O" 0 OH 10 \ / OP 0H 3 -O 0-iPr 10) An amidite allowing for introduction of an active ester in a single chemical step was prepared analogously to the method described in US Patent No. 6320041: NOONO -N(iPr) 2 0 - 00-iPr Example 4. The following is a general procedure that was used for the synthesis of pure monosubstituted PEG polymers. This method utilizes non-derivatized PEG used in excess over the selected phosphoramidite reagent in order to obtain better selectivity. Amidites containing a hydrophobic moiety, like trityl, substituted trityl, long chain fatty esters, or acetals introduce a separation handle that can be used for reversed-phase based chromatographic separation of the product. Thus PEG 6000 (60 g, 10 mmole, 2 eq.) was dried by double coevaporation with toluene (heating was applied in order to dissolve all material). The residue was dissolved in dry acetonitrile (50 mL) and an appropriate phosphoramidite (5 mmole, 1 eq.) in dry acetonitrile (30 mL) was added in one portion. To this clear solution, stirred at room temperature, a solution of 4,5-dicyanoimidazole (5.9 g, 50 mmol, 10 eq.) in acetonitrile was added in a single portion and the mixture was stirred for 15 minutes. Reaction was stopped by addition of a solution of iodine (0.1 M, 1.2 eq.) in THF:pyridine:water (10:10:1) and the brownish solution was stirred for 3 minutes. Aqueous solution of sodium bisulfite was added in small portions until decolorization 132 WO 2013/186632 PCT/IB2013/001885 of the reaction mixture. After evaporation of most of the volatile matter, the residue was dissolved in dichloromethane and extracted with a saturated aqueous solution of sodium bicarbonate. The evaporated organic phase was dried by coevaporation with toluene, and the residue was crystallized from isopropanol. The isolated crystals were dissolved in a small amount of dichloromethane and precipitated by addition of diethyl ether. The final mixture, being a composition containing a free non-derivatized PEG, mono- and bis-derivatized PEG, was preperatively purified by reverse-phase chromatography. Fractions containing the desired product were evaporated, dried and precipitated from diethyl ether. When removal of the acid labile separation handle was required the polymer is dissolved in isopropanol (with slight heating) and a 5% solution of trichloracetic acid in isopropanol was added. The mixture is cooled down in the freezer to obtain a deprotected crystalline product. If a TLC test (silicagel plates in 10% MeOH:DCM) for the purity of the product still showed the presence of the trityl protecting group, the procedure of acidic deprotection was repeated. Deprotection of other functionalities were performed following the literature procedures as far as was possible, but with some modifications: 1) Trityl protected thiol modified polymer was deprotected with a silver nitrate solution in water. After 30 minutes the solution was treated with dithiotreithiol (2 eq. to the amount of silver ions added) and the pH was raised to 9.0 by addition of potassium carbonate. After 30 minutes, the mixture was filtered through a pad of celite and extracted by dichloromethane. The organic phase was evaporated and thiolated PEG was crystallized from isopropanol. 2) Polymers modified with different aldehyde-introducing phosphoramidites were first treated with acid to remove the trityl or the acetal function. In the case of reagent 7 (Example 3) the FMOC group was removed by treatment of the polymer with 10% piperidine in DCM for 4 hours, followed by quick evaporation of the volatile matter and crystallization of the residue from isopropanol. The final conversion of the cis-amino alcohol or cis-diol to the aldehyde was done by means of 10 mM sodium periodate. Example 5. 133 WO 2013/186632 PCT/IB2013/001885 Pure, mono-substituted PEG molecules used for further derivatization were prepared as follows. Certain PEG derivatives can be prepared in a large scale and at reasonably low cost. These derivatives can be regarded by themselves as valuable modifications, but also as a good monovalent starting material, well suited for further derivatization. This Example provides for the preparation of three such derivatives: 1) Mono DMTr-O- substituted, linear PEG. An appropriate PEG (100 g, 2 eq.) was dried by double coevaporation with dry pyridine, dissolved in pyridine (100 mL), and DMTrCl (1 eq.) was added to the stirred mixture at room temperature. The yellow solution was stirred for 24 hours and methanol (10 mL) was added. The reaction mixture was stirred for an additional 60 minutes, evaporated, dissolved in dichloromethane, and treated with an aqueous solution of sodium bicarbonate. The organic phase was evaporated, dried by coevaporation with toluene, and all PEG was isolated after precipitation from diethyl ether. The collected mixture of PEG's was purified by reverse-phase chromatography. 2) Mono MMTr-NH- substituted, linear PEG. A commercial monoamino substituted PEG (1 eq.) or a material prepared according to any of existing procedures, was dried by double coevaporation with dry pyridine. The residue was dissolved in pyridine (10 mL/mmole of PEG) and trimethylchlorosilane (TMSCl) (4 eq.) was added. The mixture was stirred at room temperature for 4 hours and MMTrCl (1.5 eq.) was added. The reaction mixture was stirred overnight, and then methanol (50 eq.) was added and the mixture was stirred for an additional two hours. The total PEG was isolated after evaporation, drying by coevaporation with toluene, crystallization from isopropanol and precipitation from diethylether. The collected mixture of PEG's was purified by reverse-phase chromatography. 3) Mono Tr-S-substituted, linear PEG. An appropriate, non-derivatized PEG was dried by coevaporation with toluene and dissolved in dry DMF (5 mL/mmole). To this stirred solution at room temperature a preformed solution of phosphoroxychloride (0.4 eq.) in dry DMF (5 mL/mmole) was added and stirring was continued for 6 hours. Most of the solvent was then evaporated, and the residue was treated using a saturated aqueous sodium bicarbonate solution. Polyethylene glycol was extracted with 134 WO 2013/186632 PCT/IB2013/001885 dichloromethane and concentrated by evaporation of all volatile matter. The reaction mixture, containing the monochloro-derivatized PEG was dissolved in ethanol (10 mL/ mmole). Triphenylmethyl mercaptan (1.3 eq. to the starting PEG) was suspended in ethanol (10 mL/mmole) and converted to the sodium salt by addition of an equivalent amount of sodium hydroxide dissolved in a small amount of water. The salt was combined with the ethanolic solution of PEG and the mixture was stirred overnight. After evaporation of ethanol, the residue was partitioned between dichloromethane and aqueous sodium bicarbonate. The organic phase was evaporated and the residue was crystallized from isopropanol, followed by precipitation of total PEG from diethyl ether. The collected mixture of PEG's was purified by reverse-phase chromatography. Example 6. a) Conversion of monosubstituted PEG to bis-derivatized PEG was performed using the following method. A one equivalent of commercial methyl PEG (mPEG), (or any of partially protected PEG's from the Examples 4 or 5), was dried by coevaporation with toluene, and the polymer was dissolved in dry acetonitrile (5 mL/mmole). The modifying phosphoramidite (2-3 eq.) in dry acetonitrile (3 mL/mmole) was added, followed by a suitable activator (10-15 eq.) and the reaction mixture was stirred at RT for 15 minutes. At this point all reactions were analyzed by fast reversed-phase analytical chromatography that showed disappearance of all starting material and formation of more hydrophobic product. Reaction was quenched by addition of an oxidizing iodine solution (1.5 eq. to the amount of the phosphoramidite) or t-butylhydrogenperoxide (4 eq. to the amount of the phosphoramidite) in cases when the synthesized product did not tolerate iodine or water. The iodine-treated reaction mixtures were evaporated, dissolved in dichloromethane, decolorized with bisulfite, and the organic phase was evaporated. The t butylhydrogenperoxide treated mixtures were evaporated directly. Both mixtures were partially purified by crystallization from isopropanol, followed by precipitation from diethyl ether to obtain a pure, mono-functionalized, bis-substituted PEG. b) An azeotropically dried PEG was dissolved in dry acetonitrile (5 mL/mmole). The modifying phosphoramidite (1.0 eq.) in dry acetonitrile (3 mL/mmole) was added, followed by a 135 WO 2013/186632 PCT/IB2013/001885 suitable activator (10-15 eq.) and the reaction mixture was stirred at RT for 30 minutes. Reaction was quenched by addition of an oxidizing iodine solution (1.5 eq. to the amount of the phosphoramidite) or t-butyl hydrogenperoxide (4 eq. to the amount of the phosphoramidite) in cases when the synthesized product did not tolerate iodine or water. The iodine-treated reaction mixtures were evaporated, dissolved in dichloromethane, decolorized with bisulfite, and the organic phase was evaporated. The t-butylhydrogenperoxide treated mixtures were evaporated directly. Both mixtures were partially purified by crystallization from isopropanol, followed by precipitation from diethyl ether to obtain reaction mixture free of low molecular components. This mixture was separated on an analytical and on a preparative RP chromatographic system which allow for isolation of pure monosubstituted product. This methodology could be easy applied for amidites introducing amino, hydroxylamino, hydrazo, carboxyl, aldehyde and the biotin group. The isolated products could be deprotected upon treatment with appropriate acidic conditions, followed by precipitation of the deprotected PEG from diethylether, or the non deprotected material could be used in preparation of hetero-bis-functionalized polymer as presented below. c) Using the above idea of conversion of PEG diol into a pure, mono-functionalized product from Example 6 b) with subsequent derivatization with another reagent, the following compounds were prepared. 1) MMTr-NH-(CH 2
)
5 -O-PO-(O-iPr)O-PEG-O(0-iPr)-PO-O-(CH 2
)
6 -0-NH-MMTr 2) MMTr-NH-(CH 2
)
5 -O-PO-(O-iPr)O-PEG-O(0-iPr)-PO-0-CH(CH 3
)(CH
2
)
2 -COO-ClTr. The second derivatization were performed as in Example 6a) using excess of the second reagent over PEG to insure quantitative conversion to the double functionalized product The final compound was analyzed by HPLC, but any preparative chromatography at this stage was not needed. Example 7. The following describes methods used to introduce functional groups which were not stable in the presence of the phosphoramidite group. 136 WO 2013/186632 PCT/IB2013/001885 Diethylene glycol protected on one end with a DMTr group, and containing a H phosphonate group at the other end, was synthesized from a tritylated diol by a standard PCl 3 /triazole method according to Garegg et al., Chem. Scr. 1986, 26, 59-62. The above H-phosphonate (3 eq.) and mPEG (1 eq.) were dried by repeated coevaporation with dry pyridine. The residue was dissolved in pyridine (10 mL/mmole) and treated with pivaloyl chloride (9 eq.). The mixture was stirred at room temperature for 2 hours and the reaction was quenched by addition of triethylammonium bicarbonate (TEAB) (1 M, 5 mL/ mmole). The reaction mixture was concentrated and partitioned between dichloromethane and diluted TEAB. The organic phase was evaporated, the residue was dried by repeated coevaporation with toluene, and total PEG was purified by precipitation from diethyl ether. The isolated precipitate was filtered, dissolved in pyridine/carbon tetrachloride 2:1 and 1 -amino-6 azidohexane (4 eq.) was added. The stirred mixture was left overnight at room temperature. The mixture was evaporated, coevaporated with water, and the residual material was treated with an aqueous ammonia solution (25%, 20 mL/mmole) for 4 hours at room temperature in order to cleave the residual non-oxidized H-phosphonate dimer. Ammonia was evaporated and the resulting crude product was purified by means of preparative reverse-phase chromatography. Final removal of the DMTr group and precipitation of the PEG gave the pure azido modified product. Example 8. Conjugation of omalizumab The following describes methods used to conjugate omalizumab with a modified PEG 20K polymer. Selective DMTr protection of one end of the PEG chain. Commercial PEG 20,000 (20K) (20 g, 1 mmol) was dried by repeated co-evaporation with toluene (3x 200 mL) followed by co-evaporation with dichlormethane (200 mL). The residue was dissolved in DCM and dimethylaminopyridine DMAP (0.2 mmol), dry triethylamine (5 mm ol) and DMTrCl (1 mmol) were added. The mixture was stirred overnight at room temperature and all volatile matters were evaporated under reduced pressure. The solidified products were dissolved in a minimal volume of acetonitrile (25 mL) and cold (-20 deg C) 137 WO 2013/186632 PCT/IB2013/001885 isopropanol (200 mL) was gradually poured into this solution with magnetic stirring. The crystallized mixture of PEG derivatives was vacuum filtered, washed with cold isopropanol, and dried overnight at reduced pressure. A 3 gram portion of this material was dissolved in 10 mL of water/EtOH (4:1) and applied on a manually packed Hamilton PRP-1 Polystyrene column (3x10 cm), washed with ethanol, and equilibrated with 20 % EtOH/water containing 0.2 % conc ammonia solution. In order to ensure that all non-derivatized PEG has been eluted out from the column, elution of all products was done using a gradient of ethanol in water: Solvent A: 20 % EtOH +0.2 % ammonia; solvent B: 100 % EtOH + 0.2 % ammonia. The run was done very slowly and the starting, non-derivatized PEG came out at a solvent mixture of 22-25 % of B, while the broad peak of mono tritylated product came within a solvent mixture of 46-55 % of B. It should be noted that only the first part of this broad peak: at a solvent mixture of 46-50 % of B was used in further synthetic steps. It implies, most probably, that reverse phase (RP) separation of tritylated PEG fractionates the PEG molecules according to their molecular wight, with larger molecules coming first. All runs were performed at the flow of 2 mL/min. The collected fractions were examined for the presence of PEG using a colorimetric test using a barium chloride and KI/iodine solution. This test together with the chromatogram showing the absorbance of the DMTr group indicated that the isolated product was the mono DMTr protected PEG. This material was free of bis-tritylated product and free of non-derivatized PEG. The purity of the isolated material was confirmed by a separate HPLC analysis on a C- 18 RP column. The product was concentrated to dryness, dissolved in a minimal amount of acetonitrile (1 mL), precipitated from diethyl ether (50 mL) and dried under vacuum. Synthesis of the hetero bi-functional active ester form of PEG 0 0 0 11P- _ 0,, N-O O 0 Tr 0 iPr 138 WO 2013/186632 PCT/IB2013/001885 Mono-DMTr protected PEG (1.0 g, 0.05 mmol) was dried by double coevaporation with dry acetonitrile (2 x 30 mL) and previously synthesized amidite (See Example 3) that introduces NHS-ester function (0.25 mmol, 125 mg) was added followed by addition of tetrazol solution (0.1 M) in dry acetonitrile (13 mL, 1.3 mmol, 5 eq per amidite). 0 O O-iPr N-O1 N N(iPr) 2 0 The mixture was stirred at RT for 30 min and an oxidizing agent t-butyl hydrogen peroxide solution in dry toluene (IM, 2 mL, 8 eq) was added in a single portion. Stirring was continued for an additional 30 minutes and the volatile components were evaporated at reduced pressure. The residue was dissolved in 2 mL of acetonitrile and the non-solubilized tetrazole was spun down. The clear upper solution was transferred to a larger 50 mL Falcon tube and cold isopropanol (-20 'C) was gradually added causing crystallization of the PEG molecule. This material was spun down, washed again with cold isopropanol, and dried under vacuum (oil pump). The product was obtained in the form of white fluffy crystals. Conjugation of omalizumab to PEG reagent. Conjugation of PEG to IgG omalizumab was performed following the methodology described in J. Decruex, R. Vanbever and, P. R. Crocker, Bioconjugate Chem. (2008) 19: 2088 2094. Briefly, omalizumab (300 mg; SEQ ID NO:1) was dissolved in water (5 ml) and portionized into separate S6rsted tubes containing 15 mg of IgG each. One of the tubes was diluted with (PBS) phosphate buffer (0.2 M, pH 7.3) to 1 mL and placed on a NAP 10 desalting column previously equilibrated with the same buffer. The desalted protein was isolated with 1.5 mL of the buffer. To a portion of the desalted IgG (0.5 ml, 5 mg, 0.033 micromol), PEG reagent (20 mg, ca 5 micromol, 30 eq) was added and the mixture was left for 180 min with occasional shaking. After this time a solution of glycine (0.1 M) in phosphate buffer (0.2 mL) was added to quench any non-reacted PEG reagent. 139 WO 2013/186632 PCT/IB2013/001885 The conjugate was analyzed by the following methods: 1) SDS electrophoresis 7,5 % acrylamide gel for 90 min and 180 V. Proteins were stained with Coomassie blue (see FIG. 2). 2) The above gel was additionally stained by using a solution of barium chloride and iodine which is known to selectively label PEG containing molecules in a brown to black color dependent on the amount of PEG (this is the main method for detection of non-derivatized PEG). During this step the previously Coomassie stained proteins did not change their color (see FIG. 3). 3) SDS gel electrophoresis was also run using lower amounts of both reference IgG and conjugation reaction, and was performed for a longer time (180 min) and higher voltage (220 V) in order to achieve better conjugate separation. The gel was stained using Coomassie blue. Figure 4 shows the conjugate on the left side and free IgG on the right. 4) Using the methodology described above, two additional conjugation reactions were also performed. Both reactions were made starting from 5 mg of omalizumab in PBS buffer. For 10 equivalents excess of PEG reagent, a portion of 7.5 mg of PEG NHS was added, and for a 5 equivalents reaction, 3.75 mg PEG was added. Both reactions were quenched after 3 hours and analyzed together alongside the previous 30 equivalents reaction by SDS gel electrophoresis using 8% acrylamide. This time runs times were 180 min to achieve better separation. Figure 5 shows the results of the reactions as follows: Lane 1 - 2 tg of IgG conjugate obtained by using 5 eq of PEG reagent Lane 2 - 2 tg of IgG conjugate obtained by using 10 eq of PEG reagent Lane 3 - 2 tg of IgG conjugate obtained by using 30 eq of PEG reagent Lane 4 - 2 tg of IgG starting material Lane 5 - protein mass standards Lane 6 - 5 tg of IgG conjugate obtained by using 5 eq of PEG reagent Lane 7 - 5 tg of IgG conjugate obtained by using 10 eq of PEG reagent Lane 8 - 5 tg of IgG conjugate obtained by using 30 eq of PEG reagent 140 WO 2013/186632 PCT/IB2013/001885 Lane 9 - 5 tg of IgG starting material Lane 10 - protein mass standards 5) Analysis of the reaction mixture was also performed using gel permeation chromatography. This analysis was performed using a Zorbax GF-450 HPLC column and phosphate buffer (0.2 M, pH 7.0) as eluent using a 1.0 mL/min flow. Figure 6 shows that the pool of omalizumab conjugates was well separated from the starting non-conjugated omalizumab. Example 9. Pegylation of lysozyme and insulin 5 mg of each protein (0.35 tmol of lysozyme (Mw - 14,300) and 0.86 tmol of insulin (Mw = 5,808; SEQ ID NO.2)) were dissolved in phosphate buffer (0.2 M, pH 7.4) and PEGylating (in form of previously described NHS reagent - see Example 8) 20kD was added in 5-fold excess. The reaction was kept overnight and was quenched by addition of excess glycine. A sample of both starting protein and pegylated reaction mixtures were tested on a Zorbax GF 450 gel filtration HPLC column, with 280 nm detection, to determine the degree of derivatization. As shown in Figure 8, the normalized chromatogram shows lysozyme (dashed line) and lysozyme pegylation reaction (solid line). The double peak (at approximately 10-12 min) represents multiply pegylated lysozyme, the peak at 13 min is the residual PEG reagent, and the last peak represents unreacted protein. As shown in Figure 9, the chromatogram shows insulin (dashed line) and insulin pegylation reaction mixture (solid line). For an unknown reason, insulin (despite its formally lower Mw) has a shorter retention time than lysosome, and it co-elutes with the residual PEG reagent. The fastest running multiple peak (10-12 min) represents pegylated insulin. Example 10. Pegylation of etanercept (ENBREL@) Etanercept (SEQ ID NO:3) was pegylated in the same manner as described in Example 8. Both omalizumab and etancercept have very similar Mw, but etanercept is not a monoclonal Ab, but is instead a soluble receptor. The different shapes of these two proteins is probably the reason 141 WO 2013/186632 PCT/IB2013/001885 why in gel filtration HPLC analysis free etanercept is running closer to its pegylated conjugate than in the case of omalizumab. SDS electrophoresis 7.5 % acrylamide gel run for 90 min and at 180 V. Proteins were stained with Coomassie blue. Figure 11 shows the results of the reactions as follows: 1) Lane 8 shows pegylation of etancercept using 5 eq of PEG reagent - the residual amount of free etancercept also appears in this lane. 2) Lane 5 shows pegylation using 10 eq of PEG reagent - free etancercept is not visible any longer - at least three different bands of pegylated etancercept are shown. 3) Lane 1 shows pegylation with 30 eq of PEG reagent. All etancercept is pegylated. 4) Lane 4 is a commercial protein ladder. Figure 12 shows the normalized chromatogram showing free etanercept (dashed line), and pegylated etanercept after reaction with 10 equivalents of the pegylating reagent (solid line). During the preparative HPLC run, half minute long fractions were collected starting from 7.5 min, and ending at 10.0 minutes. The content of proteins was measured photometrically at 280 nm. To avoid all possible contamination of free etanercept in the conjugate fraction, a fraction from 8.0 min to 8.5 min was chosen for the further studies. Example 11. Evaluation of Pegylated etanercept (ENBREL@) by ELISA Material and Methods ENBREL@ (etanercept; Wyeth Europa Ltd, Berkshire, UK) was conjugated to a 20 KDa PEG (PEG20) as described in Example 8. The 20 KDa PEG was conjugated at 5 times (5 eq) or 10 times (10 eq) molar excess compared to etanercept. Recombinant human TNF-alpha (rhTNF-alpha; AbCam PLC, Cambridge, UK; at a fixed concentration of 2.9 ng/ml) was mixed with different concentrations (29000 ng/ml 56.6 pg/ml) of non-PEGylated etanercept, PEG20-etanercept (5 eq) or PEG20-etanercept (10 eq). The pre-incubation mixtures were incubated in a refrigerator at 4-80 C overnight. The amount of unbound rhTNF-alpha was analysed with a sensitive enzyme-linked immunosorbent assay (ELISA). In brief, 96-well plates (MaxiSorb, Nunc, Roskilde, Denmark) were used for the ELISA. Plates were coated with two anti-TNF-alpha monoclonal antibodies (TNF3/4, Mabtech AB, Nacka Strand, Sweden) diluted in PBS, pH 7.4, to a final concentration of 2 ig/ml. The 96-well plates were incubated in a refrigerator 142 WO 2013/186632 PCT/IB2013/001885 at 4-80 C overnight. The 96-well plates were thereafter repeatedly washed with PBS/0.05 % Tween 20. rhTNF-alpha standard (dilution series 1:1) and the pre-incubation mixture containing rhTNF-alpha/PEG20-etanercept (5 or 10 eq) or rhTNF-alpha/etanercept (non PEGylated) diluted in PBS/0.1 % BSA were thereafter added to the plates and incubated for 2 hours at room temperature. The plates were repeatedly washed with PBS/0.05 % Tween 20 and thereafter incubated 1 hour at room temperature with 1 ig/ml of a biotin conjugated anti-TNF-alpha antibody (TNF5, Mabtech AB, Nacka Strand, Sweden) diluted in PBS/0.1 % BSA/0.05 % Tween 20. Finally, streptavidine-HRP (Mabtech AB, Nacka Strand, Sweden) diluted 1:1000 in PBS/0.1 % BSA/0.05 % Tween 20 was added to the plates and incubated 1 hour following a repeated wash step with PBS/0.05 % Tween. A developer (3, 3', 5, 5' tetramethylbenzidine, TMB; Sigma-Aldrich, St Louis, MI, USA) was added after a final repeated wash with PBS/0.05 % Tween 20. A blue color reaction developed and the reaction was terminated by the addition of 1 M H 2
SO
4 15-20 minutes. The optical density of the resultant yellow color was measured in a spectrophotometer at 450 nm. The resulting ODs were plotted in GraphPad Prism 6.0 and fitted to a 4-parameter logistic curve fitting algorithm. Results The covalent attachment of PEG20 did not disturb etanercepts binding capacity of rhTNF-alpha. IC 50 values of etanercept (with or without PEG20) dilution curves were calculated and are shown in FIG. 13. The results demonstrate that the IC 50 values of PEG20-etanercept (5 or 10 eq) are less than 2-fold higher compared to non-PEGylated etanercept (FIG. 13). The IC 50 concentrations of non-PEGylated and PEGylated etanercept clearly indicate that a 20 KDa PEG does not significantly impair the binding and neutralization of TNF-a by etanercept. In addition, the use of 5 or 10 equivalents of 20 KDa PEG during the conjugation process to etanercept does not deteriorate the inhibition of TNF-a. Example 12. Biosensor Evaluation of the Binding of Pegylated Omalizumab to Human IgE 143 WO 2013/186632 PCT/IB2013/001885 The method was based on LigandTracer technology developed by RidgeView Instruments AB, Uppsala, Sweden (www.ridgeview.eu). The experiment was performed on rotated petri dishes. 2 tg of human IgE (AbCam, Cambridge, UK) was adsorbed on the plastic surface and allowed to dry over-night. 100 tg omalizumab (Genentech, CA, USA) was labeled with 125I (13.1 MBq/100 tg) according to the chloramine T method. 12 5 I-labeled omalizumab (10 nM) was then added to the IgE on the rotating petri dish. After approximately 3 1% hour the labeled antibody was exchanged with a buffer (see FIG. 14). Unlabeled omalizumab or PEG20-omalizumab (approx. 200 nM; see Example 8) was added to evaluate the ability to compete and displace 125 I-labeled omalizumab bound to IgE. The upper curve (blue) shows the binding of 125 I-labeled omalizumab to IgE without competition (FIG. 14). The lower curve (red) shows how 12 5 1-labeled omalizumab bound to IgE is displaced and competed out by unlabeled omalizumab. The curve in between (black) demonstrates that PEG20-omalizumab (30 eq) has the ability to displace and partially compete out 125 I-labeled omalizumab bound to IgE. Example 13. Synthesis of a reagent for introduction of an aminoxy group, containing a linker unit similar to the polyethylene glycol chain. o N H / MMTr -N-O O- P Monotosylate of diethylene glycol. To an ice chilled solution of diethyleneglycol (19 mL, 200 mmol) in anhydrous pyridine (8.1 mL, 100 mmol) and dichlormethane (50 ml), was added in a single portion of a solution of p-toluenesulfonyl chloride (7.6 g, 40 mmol) in dichlormethane (30 mL). The mixture was stirred at room temperature overnight and partitioned between a saturated solution of sodium bicarbonate and dichloromethane (2x200 mL). The combined organic phases 144 WO 2013/186632 PCT/IB2013/001885 were evaporated and coevaporated with toluene (2x 100 mL) to remove pyridine and traces of water. The remaining yellow oil was purified using flash silica gel chromatography, yielding the title product in the form of colorless oil in a 67% yield. N-2-(2-Hydroxyethoxy)ethoxyphtalimide. To a stirred solution of N-hydroxyphtalimide (4.3 g, 26.2 mmol) in dry DMF (mL) was added DBU (4.0 g, 26.2 mmol). To this dark red solution was added monotosylate of diethylene glycol (6.8 g, 26.2 mmol). The reaction mixture was heated at 80'C overnight, concentrated in vacuo, dissolved in ethyl acetate (200 mL) and extracted with saturated sodium bicarbonate until the aqueous phase was colorless. The organic phase was evaporated and dried by coevaporation with toluene. The title product was obtained after purification by flash silica gel chromatography as white solid. Yield 74%. Hydrazinolysis of N-2-(2-Hydroxyethoxy)ethoxyphtalimide. To a stirred solution of N-2-(2 Hydroxyethoxy)ethoxyphtalimide ( 4. 85 g, 19.3 mmol) in ethanol was added hydrazine hydrate (25 mmol) and the mixture was stirred at room temperature for 1 hr. The white solid formed during this reaction was filtered and the filtrate was evaporated to dryness and coevaporated with toluene (3x50 mL) yielding an oil, which was used further without purification. Tritylation of alkoxy amine. The oily compound obtained above was dissolved in dry pyridine (100 mL) and to this stirred solution trimethylchlorosilane (4.9 mL, 38.6 mmol) was added in one portion. The mixture was stirred for 30 min and monomethoxy trityl chloride (MMTrCl) (8.9 g, 29 mmol) was added. This mixture was stirred at room temperature overnight and was desilylated by addition of methanol (50 mL). The clear mixture was stirred for 2 hr., concentrated at reduced pressure and the residue was partitioned between saturated sodium bicarbonate and chloroform. The combined chloroform extracts (2x200 mL) were evaporated, coevaporated with toluene (2x 100 mL), and the residual oil was purified by flush silica gel chromatography, yielding the product in form of oil. Yield 77%. Synthesis of the title phosphoramidite. The N-MMTr protected aminoxy alcohol (5.55 g, 14,8 mmol) was dried by coevaporation with toluene (2x50 mL), dissolved in dry dichloromethane (100 mL) and dry triethylamine (6.0 mL, 59.2 mmol) was added followed by isopropyl, N,N diisopropylphosphoramidochloridite (22.2 mmol). The rest of this reaction was made in accordance with the general description of phosphitylation process as in Example 3. The final product was obtained in 81 % yield in the form of a slightly yellow oil. 145 WO 2013/186632 PCT/IB2013/001885 Example 14 This example provides a process for preparing a reagent which introduces an aliphatic aldehyde group to a previously monofunctionalized polyethylene glycol (e.g., a DMTr containing polymer). The fully derivatized PEG is deprotected in a single step upon treatment with diluted hydrochloric acid. MeO N Me O-P 2-(2,2-Dimethoxy-ethoxy) -ethanol. This compound was obtained from ethylene glycol and 2-chloro- 1,1 -dimethoxy-ethane following procedure described by Lei Tao et al. J. Am. Chem. Soc. 2004, 126, 13220-13221. The final product was taken to the following reaction without purification. The hydroxyl acetal was converted to the title product following the methodology from Example 3, yielding, upon silica gel purification the product in a 61 % yield as colorless oil. 1) An essentially pure, modified polymer, comprising a water soluble, non peptidic and non nucleotidic, linear or branched polymer backbone, containing from 2 and up to 100 termini, and having at least one terminus being covalently bonded to the structure: E R1
A-O-P-Z
1 -L-M-R
Z
2 K-G wherein: 146 WO 2013/186632 PCT/IB2013/001885 A is the point of bonding to the terminus of the polymer backbone; E is oxygen or sulfur; K is selected from the group consisting of linear alkyl, branched alkyl, alkyloxyalkyl, or oligomeric form of alkyloxyalkyl; G is none, or is selected from the group consisting of an alkoxy, trityloxy or substituted trityloxy group;
Z
1 and Z 2 are 0 or NH in such a way that both Z1 and Z 2 may be 0, but when ZI is NH then Z 2 is 0, and when Z 2 is NH then Z 1 is 0. L is selected from the group consisting of linear alkyl, branched alkyl, nucleoside, alkyloxyalkyl, oligomeric form of alkyloxyalkyl, aryl and substituted aryl; M is a group reactive with a biologically active molecule or detectable functional group; R is selected from a group consisting of protecting groups, hydrophobic separation handles, activating groups, hydrogen or none, R 1 is a hydrophobic separation handle or none, and both R and R 1 can coexist providing that there is only one hydrophobic separation handle within the molecule or the hydrophobicity of either of these groups is substantially higher than the other; 2) The modified polymer of claim 1, wherein the functional group M is selected from the group consisting of hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide, biotin or a fluorophore. 3) The modified polymer of claim 1, wherein the group K is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, hexyl, or a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 4) The modified polymer of claim 1, wherein the group L is CI-C 1 2 alkyl or substituted alkyl. 5) The modified polymer of claim 1, wherein the group R is selected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, FMOC, trifluoroacetyl, acetal, cyclic acetal or combinations thereof. 6) The modified polymer of claim 1, wherein, for the functional group M being carboxyl, the group R is selected from the group consisting of chlorotrityl, trityl, N 147 WO 2013/186632 PCT/IB2013/001885 hydroxysuccinimidyl, p-nitrophenyl, pentachlorophenyl, simple non-activating alkyls selected from the group of C 1 -Cis alkyls or none. 7) The modified polymer of claim 1, wherein the non peptidic and non nucleotidic polymer backbone is selected from the group consisting of poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol), polyoxazoline, and copolymers and mixtures thereof. 8) The modified polymer of claim 1, wherein the non peptidic and non nucleotidic polymer backbone is poly(ethylene glycol). 9) The modified polymer of claim 8, wherein the poly(ethylene glycol) has an average molecular weight from about 500 Da to about 100000 Da. 10) An essentially pure, linear form of modified polymer of claim 1, wherein the two termini of the polymer are modified non-symmetrically with two different functional groups and wherein the two functional groups are linked to the polymer as in the structure: E1 E
R
1
-M
1
-L
1
-Z
1 -P-O polymer - -P-Z 3 -L-M-R I I Z2 Z4
G
1
-K
1 K-G wherein: M and Mi are two different functional groups reactive with a biologically active molecule or detectable functional groups; E and E 1 are independently oxygen or sulfur; The pair Z 1 and Z 2 are independent from the pair Z 3 and Z 4 and both Z1 and
Z
2 may be 0, but when Z 1 is NH then Z 2 is 0, and when Z 2 is NH then Z 1 is 0, similarly for a pair of Z 3 and Z 4 - both Z 3 and Z 4 may be 0, but when Z 3 is NH then
Z
4 is 0, and when Z 4 is NH then Z 3 is 0; L and Li are independently selected from the group consisting of linear alkyl, branched alkyl, alkyloxyalkyl, oligomeric form of alkyloxyalkyl, aryl and substituted aryl; R and R 1 are independently protecting groups, activating groups or none; K and K 1 are independently selected from the group consisting of linear alkyl, branched alkyl, alkyloxyalkyl, or oligomeric form of alkyloxyalkyl; G and G 1 are independently selected from the group consisting of none, an alkoxy, trityloxy or substituted trityloxy group; 148 WO 2013/186632 PCT/IB2013/001885 L-M-R and L 1
-M
1
-R
1 fragments are linked to the respective terminus of the said polymer via phosphotriester, thiophosphotriester or amidophosphotriester bonds; 11) The modified polymer of claim 10, wherein the two different functional groups M and Mi are selected independently from the group consisting of hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal,dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide, biotin or a fluorophore. 12) The modified polymer of claim 10, wherein the group K is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, hexyl, or a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 13) The modified polymer of claim 10, wherein the group L is C 1
-C
1 2 alkyl or substituted alkyl. 14) The modified polymer of claim 10, wherein the group R is selected from trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, FMOC, trifluoroacetyl, acetal, cyclic acetal or combinations of thereof. 15) The modified polymer of claim 10, wherein, for the functional group M being carboxyl, the group R is selected from the group consisting of chlorotrityl, trityl, N hydroxysuccinimidyl, p-nitrophenyl, pentachlorophenyl, simple non-activating alkyls selected from the group of C 1 -CIs alkyls or none. 16) The modified polymer of claim 10, wherein the polymer is poly(ethylene glycol). 17) The modified polymer of claim 16, wherein the poly(ethylene glycol) has an average molecular weight from about 500 Da to about 100000 Da. 18) An essentially pure, linear form of modified polymer of claim 1, wherein the two termini of the polymer are modified non-symmetrically with two different functional groups and wherein the two functional groups are linked to the polymer as in the structure: E R2--M2 polymer O- P-Z 1 - L- M--R
Z
2 K-G wherein: M and M 2 are two different functional groups reactive with a biologically active molecules or M is a detectable functional group; E is O or S; 149 WO 2013/186632 PCT/IB2013/001885 K is selected from the group consisting of linear alkyl, branched alkyl, alkyloxyalkyl, or oligomeric form of alkyloxyalkyl; G is none or is selected from the group consisting of an alkoxy, trityloxy, monoalkoxy substituted trityloxy group or dialkoxy substityted trityloxy group;
Z
1 and Z 2 are 0 or NH in such way that both ZI and Z 2 may be 0, but when
Z
1 is NH then Z 2 is 0, and when Z 2 is NH then Z 1 is 0; L is selected from the group consisting of linear alkyl, branched alkyl, alkyloxyalkyl, oligomeric form of alkyloxyalkyl, aryl and substituted aryl; R is a protecting group, activating group, hydrogen or none; L-M-R fragment is linked to the first terminus of the said polymer via phosphotriester, thiophosphtriester or amidophosphotriester;
M
2 is 0, S or NH;
M
2 is linked directly to the second terminus of the polymer, and not via phosphotriester, thiophosphtriester or amidophosphotriester;
R
2 is a protecting group or none. 19) The modified polymer of claim 18, wherein the functional group M is selected from the group consisting of hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal,dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide or biotin. 20) The modified polymer of claim 18, wherein the group R 2 is selected from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, FMOC, alkylcarboxyl, benzoyl, tetrahydropyranyl, methyl or none. 21) The modified polymer of claim 18, wherein the polymer is poly(ethylene glycol). 22) The conjugate of any polymer of claims 1 to 18 with a biologically active molecule wherein said biologically active molecule is selected from the group consisting of enzymes, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides and low molecular weight drugs. 23) A method of synthesizing a substantially pure, water soluble polymer of claim 1, said method comprising the steps of: a) contacting the water soluble polymer, of non-peptidic and non-nucleotidic type, and having linear or branched polymer backbone, and containing from 2 and up to 100 termini, in a water free solvent, with a selected modifying reagent in form of the phosphoramidite derivative as in the structure: 150 WO 2013/186632 PCT/IB2013/001885
R
3 N-P-0-L-M-R R4 0 K-G wherein:
R
3 and R 4 are isopropyls or are a part of of morpholine ring. b) starting reaction by addition of an activating reagent. c) oxidation of p 3 to p 5 by addition of an oxidizing reagent. d) optional chromatographic purification of the protected polymer. e) removal of the protecting groups. 24) Method of claim 23 wherein the activating reagent is selected from tetrazole, 2 ethylthiotetrazole, 2-bezylthiotetrazole, 4,5-dicyanoimidazole, "Activator 42", pyridinium hydrochloride or pyridinium trifluoroacetate 25) Method of claim 23 wherein the oxidizing reagent is selected from a group consisting of iodine, water peroxide, t-butyl hydrogen peroxide, acetone peroxide, sulfur and thiuram disulfide. 26) Method of claim 23, wherein the ratio between the polymer and the phosphoramidite is from 1:1 to 10:1 in order to facilitate formation of monosubstituted product. 27) A method of synthesizing a substantially pure, water soluble polymer of claim 10, said method comprising the steps of: a) reacting the water soluble polymer, of non-peptidic and non-nucleotidic type, having linear polymer backbone, and containing two reactive termini, in a water free solvent, with a selected first modifying reagent in form of the phosphoramidite derivative, under conditions that facilitate formation of monoderivatized product. b) chromatographic isolation of the monoderivatized polymer. c) reacting the monoderivatized product with a second modifying reagent in the form of phosphoramidite derivative, under conditions that facilitate the quantitative conversion to the double modified polymer. d) isolation of the double modified polymer by precipitation or crystallization. 28) A method of synthesizing a substantially pure, water soluble polymer of claim 18, said method comprising the steps of: a) reacting the substantially pure, linear polymer, substituted at the first terminus with a function R 2
-M
2 , with a selected modifying reagent in form of the phosphoramidite derivative under conditions facilitating the quantitative conversion of the mono substituted polymer to the double modified polymer. b) isolation of the double modified polymer by precipitation or crystallization. 151 WO 2013/186632 PCT/IB2013/001885 29) The use of any material of claim 1 to claim 28 for formation of a conjugate between this material and a biologically active molecule, wherein said biologically active molecule is selected from the group consisting of enzymes, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides and low molecular weight drugs. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 152

Claims (1)

  1. WHAT IS CLAIMED IS:
    A method of making a TNF inhibitor conjugate, said method comprising reacting a TNF inhibitor or a derivative thereof with a preparation comprising a water-soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (1) under conditions suitable for group M or R to react with a TNF inhibitor or said derivative thereof:
    or a salt thereof,
    wherein:
    A is the point of covalent bonding to the terminus of the polymer backbone;
    E is O or S;
    K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    oligomeric alkyleneoxyalkylene;
    G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    separation handle;
    Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 can be NH;
    L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene;
    M is a protected group that when deprotected is reactive with a TNF inhibitor or
    derivative thereof or is a group reactive with a TNF inhibitor or derivative thereof; R is absent or selected from the group consisting of: hydrogen, a protecting group, a hydrophobic separation handle, or an activating group;
    R1 is absent or a hydrophobic separation handle;
    wherein when M is a protected group that when deprotected is reactive with a TNF inhibitor or derivative thereof, then R is a protecting group or a hydrophobic separation handle;
    wherein when M is a group reactive with a TNF inhibitor or derivative thereof, R is absent, hydrogen, or an activating group; and
    wherein only one of R, R1, and G can be a hydrophobic separation handle, . 2. The method of claim 1, wherein the polymer has from 2 to 100 termini. 3. The method of claim 1 or claim 2, wherein only one termini of the polymer backbone is covalently bonded to the structure of formula (1). 4. The method of any one of claims 1 to 3 wherein the polymer backbone has two
    termini. 5. The method of claim 4, wherein only one termini of the polymer backbone is
    covalently bonded to the structure of formula (1). 6. The method of claim 4, wherein both termini of the polymer backbone are covalently bonded to the structure of formula (1). 7. The method of any one of claims 1 to 6, wherein one of Z1 and Z2 is NH and the other is O. 8. The method of claim 7, wherein Z1 is O and Z2 is NH. 9. The method of claim 7, wherein Z1 is NH and Z2 is O.
    10. The method of claim 7, wherein both Z1 and Z2 are O. 11. The method of any one of claims 1 to 10, wherein said group reactive with a TNF inhibitor or said derivative is selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide. 12. The method of any one of claims 1 to 11, wherein K is selected from the group
    consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 13. The method of any one of claims 1 to 12, wherein G is a substituted or unsubstituted trityloxy. 14. The method of any one of claims 1 to 13, wherein L is a substituted or unsubstituted C1-C12 alkylene. 15. The method any one of claims 1 to 14, wherein R is selected from the group
    consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal. 16. The method of any one of claims 1 to 15, wherein said group reactive with a TNF inhibitor or said derivative is carboxyl and R is absent or selected from the group consisting of: N-hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl. 17. The method of any one of claims 1 to 16, wherein said polymer backbone is selected from the group consisting of poly(alkylene glycol), poly(oxyethylated polyol), 83 poly(olefmic alcohol), poly(a -hydroxy acid), poly(vinyl alcohol), polyoxazoline, and
    84 copolymers.
    85
    86 18. The method of any one of claims 1 to 17, wherein said polymer backbone is
    87 poly(ethylene glycol).
    88
    89 19. The method of claim 18, wherein said poly(ethylene glycol) has an average molecular
    90 weight from about 500 Da to about 100,000 Da.
    91
    92 20. The method of any one of claims 1-19, wherein said preparation comprises at least
    93 50% by weight of said water-soluble, non-peptidic, and non-nucleotidic polymer
    94 backbone having at least one terminus covalently bonded to a structure of formula
    95 (1).
    96
    97 21. The method of claim 20, wherein said preparation comprises at least 98% by weight
    98 of said water-soluble, non-peptidic, and non-nucleotidic polymer backbone having at
    99 least one terminus covalently bonded to a structure of formula (1).
    100
    101 22. The method of any one of claims 1 to 21, said method further comprising (i)
    102 removing the hydrophobic separation handle(s) from said structure of formula (1) and
    103 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    104 before reacting with a TNF inhibitor or said derivative.
    105
    106 23. The method of any one of claims 1 to 21, said method further comprising removing
    107 the hydrophobic separation handle(s) from said structure of formula (1) after reacting
    108 with a TNF inhibitor or said derivative.
    109
    1 10 24. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a water-
    1 1 1 soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one
    1 12 terminus covalently bonded to a structure of formula (9): R A O 1 L L2 B
    or a salt thereof,
    wherein:
    A is the point of covalent bonding to the terminus of the polymer backbone;
    E is O or S;
    K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    oligomeric alkyleneoxyalkylene;
    G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    separation handle;
    Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 can be NH;
    L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene;
    R1 is absent or a hydrophobic separation handle, wherein only one of R1 and G can be a hydrophobic separation handle;
    L2 is a covalent linking moiety between L on the polymer backbone and B; and B is a TNF inhibitor or a derivative thereof. 25. A method of making a TNF inhibitor conjugate, said method comprising reacting a TNF inhibitor or a derivative thereof with a preparation comprising a compound of formula (2) under conditions suitable for group M or R to react with a TNF inhibitor or said derivative thereof: R1 1 L1 Z3 P O polymer O P Z1 L M R
    G1 K1 \ K G
    136 (2) 137 or a salt form thereof,
    138 wherein:
    139 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    140 backbone, wherein each linking group is bonded at a different terminus of said 141 polymer;
    142 E and E1 are independently O or S;
    143 K and Ki are independently selected from the group consisting of: alkylene, 144 alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    145 G and Gi are independently absent or are selected from the group consisting of: 146 alkoxy and a hydrophobic separation handle;
    147 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH, 148 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    149 L and L1 are independently selected from the group consisting of: a divalent radical of 150 a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and 151 unsubstituted and substituted arylene;
    152 M and M1 are independently selected from a protected group that when deprotected is 153 reactive with a TNF inhibitor or said derivative or a group reactive with a TNF 154 inhibitor or said derivative, wherein M and M1 are different; and
    155 R and R1 are independently absent, hydrogen, a protecting group, or an activating 156 group;
    157 wherein when M is a protected group that when deprotected is reactive with a TNF 158 inhibitor or said derivative, then R is a protecting group or a hydrophobic
    159 separation handle; 160 wherein when M is a group reactive with a TNF inhibitor or said derivative, R is
    161 absent, hydrogen, or an activating group;
    162 wherein when M1 is a protected group that when deprotected is reactive with a TNF
    163 inhibitor or said derivative, then R1 is a protecting group or a hydrophobic
    164 separation handle; and
    165 wherein when M1 is a group reactive with a TNF inhibitor or said derivative, R1 is
    166 absent, hydrogen, or an activating group.
    167
    168 26. The method of claim 25, wherein one of Z1 and Z2 is NH and the other is O.
    169
    170 27. The method of claim 26, wherein Z1 is O and Z2 is NH.
    171
    172 28. The method of claim 26, wherein Z1 is NH and Z2 is O.
    173
    174 29. The method of claim 25, wherein Z1 is O and Z2 is O.
    175
    176 30. The method of claim 25, wherein one of Z3 and Z4 is NH and the other is O.
    177
    178 31. The method of claim 30, wherein Z3 is O and Z4 is NH.
    179
    180 32. The method of claim 30, wherein Z3 is NH and Z4 is O.
    181
    182 33. The method of claim 25, wherein Z3 is O and Z4 is O.
    183
    184 34. The method of any one of claims 25 to 33, wherein said group reactive with a TNF
    185 inhibitor or said derivative thereof is selected from the group consisting of: hydroxyl,
    186 amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    187 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    188 iodoacetamide.
    189
    190 35. The method of any one of claims 25 to 34, wherein K or K1 is independently selected
    191 from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene,
    192 isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene
    193 glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    194
    195 36. The method of any one of claims 25 to 35, wherein G or G1 is independently a
    196 substituted or unsubstituted trityloxy.
    197
    198 37. The method of any one of claims 25 to 36, wherein L or L1 is independently a
    199 substituted or unsubstituted C 1 -C 12 alkylene .
    200
    201 38. The method of any one of claims 25 to 37, wherein R or R1 is independently selected
    202 from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    203 alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and
    204 combinations of thereof.
    205
    206 39. The method of any one of claims 25 to 38, wherein said polymer is poly(ethylene
    207 glycol).
    208
    209 40. The method of claim 39, wherein said poly(ethylene glycol) has an average
    210 molecular weight from about 500 Da to about 100,000 Da.
    21 1
    212 41. The method of any one of claims 25 to 40, wherein said preparation comprises at least
    213 50% by weight of said compound.
    214
    215 42. The method of any one of claims 25 to 41, said method further comprising (i)
    216 removing the hydrophobic separation handle(s) from said structure of formula (2) and
    217 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    218 before reacting with a TNF inhibitor or said derivative.
    219
    220 43. The method of any one of claims 25 to 41, said method further comprising removing
    221 the hydrophobic separation handle(s) from said structure of formula (2) after reacting
    222 with a TNF inhibitor or said derivative.
    223
    224 44. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a structure of
    225 formula (10):
    E1 E B1 L3 L1 Z3 P O polymer O P Z1 L L2 B
    Z4 Z2
    G 11 K1 \ K G
    226 (10)
    227 or a salt form thereof,
    228 wherein:
    229 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic
    230 polymer backbone, wherein each linking group is bonded at a different terminus
    231 of said polymer;
    232 E and E1 are independently O or S;
    233 K and Ki are independently selected from the group consisting of:
    234 alkylene, alkyleneoxyalkylene, and oligomeric alky leneoxy alky lene;
    235 G and Gi are independently absent or are selected from the group
    236 consisting of: alkoxy and a hydrophobic separation handle;
    237 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O
    238 and NH, wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    239 L and L1 are independently selected from the group consisting of: a
    240 divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric
    241 alkyleneoxyalkylene, and unsubstituted and substituted arylene;
    242 L2 is a covalent linking moiety between L on the polymer backbone and
    243 B; 244 L3 is a covalent linking moiety between L on the polymer backbone and
    245 B1; and
    246 B and B1 are independently a TNF inhibitor, a derivative of a TNF
    247 inhibitor, a biologic other than a TNF inhibitor, a drug, a detectable group, a
    248 separation moiety, wherein at least one of B and B1 is a TNF inhibitor or a
    249 derivative of a TNF inhibitor.
    250
    251 45. A method of making a TNF inhibitor conjugate, said method comprising reacting a
    252 TNF inhibitor or a derivative thereof with a preparation comprising a compound of
    253 formula (3):
    E
    R2 M2 polymer O P Z L M R
    254 K G (3)
    255 or a salt form thereof,
    256 wherein:
    257 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    258 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    259 at a different terminus of said polymer;
    260 E and E1 are independently O or S;
    261 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    262 oligomeric alkyleneoxyalkylene;
    263 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    264 separation handle;
    265 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    266 can be NH;
    267 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    268 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    269 substituted arylene; 270 M is selected from a protected group that when deprotected is reactive with a TNF
    271 inhibitor or said derivative or a group reactive with a TNF inhibitor or said
    272 derivative;
    273 M2 is selected from O, S or NH; and
    274 R is absent, a protecting group, a hydrophobic separation handle, or an activating
    275 group;
    276 R2 is hydrogen or a protecting group;
    277 wherein when M is a protected group that when deprotected is reactive with a TNF
    278 inhibitor or said derivative, then R is a protecting group or a hydrophobic
    279 separation handle; and
    280 wherein when M is a group reactive with a TNF inhibitor or said derivative, R is
    281 absent, hydrogen, or an activating group.
    282
    283 46. The method of claim 45, wherein one of Z1 and Z2 is NH and the other is O.
    284
    285 47. The method of claim 45 or claim 46, wherein G is a substituted or unsubstituted
    286 trityloxy group.
    287
    288 48. The method of claim 45 or claim 46, wherein G is selected from a monoalkoxy
    289 substituted trityloxy group or dialkoxy substituted trityloxy group.
    290
    291 49. The method of any one of claims 45 to 48, wherein said group reactive with a TNF
    292 inhibitor or said derivative is selected from the group consisting of: hydroxyl, amine,
    293 thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    294 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    295 iodoacetamide.
    296 50. The method of any one of claims 45 to 49, wherein R2 is absent or selected from the
    297 group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    298 fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl, methyl. 299
    300 51. The method of any one of claims 45 to 50, wherein said polymer is poly(ethylene
    301 glycol).
    302
    303 52. The method of any one of claims 45 to 51 , wherein said preparation comprises at least
    304 50% of said compound.
    305
    306 53. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a compound of
    307 formula (
    308
    309 or a salt form thereof,
    310 wherein:
    31 1 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    312 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    313 at a different terminus of said polymer;
    314 E and E1 are independently O or S;
    315 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    316 oligomeric alkyleneoxyalkylene;
    317 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    318 separation handle;
    319 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    320 can be NH;
    321 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    322 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    323 substituted arylene;
    324 L2 is a covalent linking moiety between L on the polymer backbone and B;
    325 L4 is a covalent linking moiety between L on the polymer backbone and B1; and 326 B and B1 are independently a TNF inhibitor, a derivative of a TNF inhibitor, a
    327 biologic other than a TNF inhibitor, a drug, a detectable group, a separation
    328 moiety, wherein at least one of B and B1 is a TNF inhibitor or a derivative of a
    329 TNF inhibitor.
    330
    331 54. A method of preparing a compound comprising a water-soluble, non-peptidic, and
    332 non-nucleotidic polymer backbone having at least one terminus covalently bonded to
    333 a structure of formula (9):
    334
    E R A O P Z L L2 B
    335 Y (9)
    336 or a salt thereof,
    337 wherein:
    338 A is the point of covalent bonding to the terminus of the polymer backbone;
    339 E is O or S;
    340 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    341 heterocyclyl, aryl, and heteroaryl;
    342 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    343 can be NH;
    344 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    345 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    346 substituted arylene;
    347 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    348 B is a TNF inhibitor or a derivative thereof;
    349 comprising the steps of:
    350 (a) providing a composition comprising a compound of formula (8):
    351
    353 wherein:
    354 A is the point of covalent bonding to the terminus of the polymer backbone;
    355 E is O or S;
    356 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    357 heterocyclyl, aryl, and heteroaryl;
    358 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    359 can be NH;
    360 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    361 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    362 substituted arylene;
    363 M is a protected group that when deprotected is reactive with a TNF inhibitor or a
    364 derivative thereof;
    365 R is a hydrophobic separation handle;
    366 R1 is absent or a hydrophobic separation handle; and
    367 (b) removing the hydrophobic separation handle(s);
    368 (c) optionally reacting the compound obtained in step (b) with an activating agent;
    369 and
    370 (d) reacting the compound obtained in step (b), or, optionally in step (c), with a TNF
    371 inhibitor or a derivative thereof.
    372
    373 55. The method of claim 54, wherein Z1 is O and Z2 is NH.
    374
    375 56. The method of claim 54, wherein Z1 is NH and Z2 is O.
    376
    377 57. The method of claim 54, wherein both Z1 and Z2 are O. 378
    379 58. The method of any one of claims 54 to 57, wherein the protected group M, when
    380 deprotected, is represented by hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal,
    381 dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide,
    382 aminoxy, maleimide, dithiopyridine, or iodoacetamide.
    383
    384 59. The method of any one of claims 54 to 58, wherein R is selected from the group
    385 consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    386 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    387
    388 60. The method of any one of claims 54 to 59, wherein said polymer backbone is selected
    389 from the group consisting of poly(alkylene glycol), poly(oxyethylated polyol),
    390 poly(olefinic alcohol), poly(a -hydroxy acid), poly(vinyl alcohol), polyoxazoline, and
    391 copolymers.
    392
    393 61. The method of any one of claims 54 to 60, wherein said polymer backbone is
    394 poly(ethylene glycol).
    395
    396 62. The method of any one of claims 54 to 61, wherein reaction step (d) is carried out in
    397 the presence of water or a protic solvent.
    398
    399 63. The method of any one of claims 54 to 62, wherein the compound of formula (8)
    400 comprises at least 98% by weight of said composition.
    401
    402 64. A composition comprising the conjugate of any one of claims 24, 44, and 53, and a
    403 pharmaceutically acceptable excipient.
    404
    405 65. The composition of claim 64, wherein the polymer backbone of said conjugate is
    406 poly(ethylene glycol).
    407
    408 66. The composition of claim 64 or claim 65, wherein B is a TNF inhibitor.
    409
    410 67. The composition of any one of claims 64 to 66, wherein B1 is a TNF inhibitor.
    41 1
    412 68. A method of treating a patient diagnosed with an inflammatory disease, said method
    413 comprising administering to said patient an effective amount of the conjugate of any
    414 one of claims 24, 44, or 53.
    415
    416 69. The method of claim 68, wherein the polymer backbone of said conjugate is
    417 poly(ethylene glycol).
    418
    419 70. The method of claim 68 or claim 69, wherein B is a TNF inhibitor.
    420
    421 71. The method of any one of claims 68 to 70, wherein B1 is a TNF inhibitor.
    422
    423 72. A method of making an insulin conjugate, said method comprising reacting insulin or
    424 a derivative thereof with a preparation comprising a water-soluble, non-peptidic, and
    425 non-nucleotidic polymer backbone having at least one terminus covalently bonded to
    426 a structure of formula (1) under conditions suitable for group M or R to react with
    427 insulin or said derivative thereof:
    428
    430 or a salt thereof,
    431 wherein:
    432 A is the point of covalent bonding to the terminus of the polymer backbone; 433 E is O or S;
    434 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    435 oligomeric alkyleneoxyalkylene;
    436 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    437 separation handle;
    438 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    439 Z2 can be NH;
    440 L is selected from the group consisting of: a divalent radical of a nucleoside,
    441 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    442 substituted arylene;
    443 M is a protected group that when deprotected is reactive with insulin or derivative
    444 thereof or is a group reactive with insulin or derivative thereof;
    445 R is absent or selected from the group consisting of: hydrogen, a protecting group,
    446 a hydrophobic separation handle, or an activating group;
    447 R1 is absent or a hydrophobic separation handle;
    448 wherein when M is a protected group that when deprotected is reactive with a
    449 insulin or derivative thereof, then R is a protecting group or a hydrophobic separation
    450 handle;
    451 wherein when M is a group reactive with insulin or derivative thereof, R is absent,
    452 hydrogen, or an activating group; and
    453 wherein only one of R, R1, and G can be a hydrophobic separation handle.
    454
    455 73. The method of claim 72, wherein the polymer has from 2 to 100 termini.
    456
    457 74. The method of claim 72 or claim 73, wherein only one termini of the polymer
    458 backbone is covalently bonded to the structure of formula (1).
    459
    460 75. The method of any one of claims 73 to 74 wherein the polymer backbone has two
    461 termini.
    462
    463 76. The method of claim 75, wherein only one termini of the polymer backbone is
    464 covalently bonded to the structure of formula (1).
    465
    466 77. The method of claims 75, wherein both termini of the polymer backbone are
    467 covalently bonded to the structure of formula (1).
    468
    469 78. The method of any one of claims 72 to 77, wherein one of Z1 and Z2 is NH and the
    470 other is O.
    471
    472 79. The method of claim 78, wherein Z1 is O and Z2 is NH.
    473
    474 80. The method of claim 78, wherein Z1 is NH and Z2 is O.
    475
    476 81. The method of claim 78, wherein both Z1 and Z2 are O.
    477
    478 82. The method of any one of claims 72 to 81, wherein said group reactive with insulin or
    479 said derivative is selected from the group consisting of: hydroxyl, amine, thiol,
    480 carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide,
    481 vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide.
    482
    483 83. The method of any one of claims 72 to 82, wherein K is selected from the group
    484 consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene,
    485 sec-butylene, tert-butylene, and hexylene, or a residue from diethylene glycol,
    486 triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    487
    488 84. The method of any one of claims 72 to 83, wherein G is a substituted or unsubstituted
    489 trityloxy.
    490
    491 85. The method of any one of claims 72 to 84, wherein L is a substituted or unsubstituted
    492 C1-C12 alkylene. 493
    494 86. The method any one of claims 72 to 85, wherein R is selected from the group
    495 consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    496 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    497
    498 87. The method of any one of claims 72 to 86, wherein said group reactive with insulin or
    499 said derivative is carboxyl and R is absent or selected from the group consisting of:
    500 N-hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl.
    501 88. The method of any one of claims 1 to 16, wherein said polymer backbone is selected
    502 from the group consisting of poly(alkylene glycol), poly(oxyethylated polyol),
    503 poly(olefinic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol), polyoxazoline, and
    504 copolymers.
    505
    506 89. The method of any one of claims 72 to 88, wherein said polymer backbone is
    507 poly(ethylene glycol).
    508
    509 90. The method of claim 89, wherein said poly(ethylene glycol) has an average molecular
    510 weight from about 500 Da to about 100,000 Da.
    51 1
    512 91. The method of any one of claims 72 to 90, wherein said preparation comprises at least
    513 50% by weight of said water-soluble, non-peptidic, and non-nucleotidic polymer
    514 backbone having at least one terminus covalently bonded to a structure of formula
    515 (1).
    516
    517 92. The method of claim 91, wherein said preparation comprises at least 98% by weight
    518 of said water-soluble, non-peptidic, and non-nucleotidic polymer backbone having at
    519 least one terminus covalently bonded to a structure of formula (1).
    520
    521 93. The method of any one of claims 72 to 92, said method further comprising (i)
    522 removing the hydrophobic separation handle(s) from said structure of formula (1) and 523 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    524 before reacting with insulin or said derivative.
    525
    526 94. The method of any one of claims 72 to 92, said method further comprising removing
    527 the hydrophobic separation handle(s) from said structure of formula (1) after reacting
    528 with insulin or said derivative.
    529
    530 95. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a water-
    531 soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one
    532 terminus covalently bonded to a structure of formula (9):
    R1
    A O 1 L L2 B
    533
    534 or a salt thereof,
    535 wherein:
    536 A is the point of covalent bonding to the terminus of the polymer backbone;
    537 E is O or S;
    538 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    539 oligomeric alkyleneoxyalkylene;
    540 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    541 separation handle;
    542 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    543 Z2 can be NH;
    544 L is selected from the group consisting of: a divalent radical of a nucleoside,
    545 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    546 substituted arylene; 547 R1 is absent or a hydrophobic separation handle, wherein only one of R1 and G
    548 can be a hydrophobic separation handle;
    549 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    550 B is insulin or a derivative thereof.
    551
    552 96. A method of making an insulin conjugate, said method comprising reacting insulin or
    553 a derivative thereof with a preparation comprising a compound of formula (2) under
    554 conditions suitable for group M or R to react with insulin or said derivative thereof:
    R1 1 L1 Z3 P 11 O polymer O P \\ Z1 L M R
    G1 K1 K G
    555
    556 (2)
    557 or a salt form thereof,
    558 wherein:
    559 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    560 backbone, wherein each linking group is bonded at a different terminus of said polymer;
    561 E and E1 are independently O or S;
    562 K and Ki are independently selected from the group consisting of: alkylene,
    563 alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    564 G and Gi are independently absent or are selected from the group consisting of:
    565 alkoxy and a hydrophobic separation handle;
    566 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    567 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    568 L and L1 are independently selected from the group consisting of: a divalent
    569 radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene,
    570 and unsubstituted and substituted arylene; 571 M and M1 are independently selected from a protected group that when
    572 deprotected is reactive with insulin or said derivative or a group reactive with insulin or
    573 said derivative, wherein M and M1 are different; and
    574 R and R1 are independently absent, hydrogen, a protecting group, or an activating
    575 group;
    576 wherein when M is a protected group that when deprotected is reactive with
    577 insulin or said derivative, then R is a protecting group or a hydrophobic separation
    578 handle;
    579 wherein when M is a group reactive with insulin or said derivative, R is absent,
    580 hydrogen, or an activating group;
    581 wherein when M1 is a protected group that when deprotected is reactive with
    582 insulin or said derivative, then R1 is a protecting group or a hydrophobic separation
    583 handle; and
    584 wherein when M1 is a group reactive with insulin or said derivative, R1 is absent,
    585 hydrogen, or an activating group.
    586
    587 97. The method of claim 96, wherein one of Z1 and Z2 is NH and the other is O.
    588
    589 98. The method of claim 97, wherein Z1 is O and Z2 is NH.
    590
    591 99. The method of claim 97, wherein Z1 is NH and Z2 is O.
    592
    593 100. The method of claim 96, wherein Z1 is O and Z2 is O.
    594
    595 101. The method of claim 96, wherein one of Z3 and Z4 is NH and the other is O. 596
    597 102. The method of claim 101, wherein Z3 is O and Z4 is NH.
    598
    599 103. The method of claim 101, wherein Z3 is NH and Z4 is O.
    600
    601 104. The method of claim 96, wherein Z3 is O and Z4 is O.
    602
    603 105. The method of any one of claims 96 to 104, wherein said group reactive with
    604 insulin or said derivative thereof is selected from the group consisting of: hydroxyl,
    605 amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    606 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    607 iodoacetamide.
    608
    609 106. The method of any one of claims 96 to 105, wherein K or K1 is independently
    610 selected from the group consisting of: methylene, ethylene, propylene, isopropylene,
    61 1 butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from
    612 diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 613
    614 107. The method of any one of claims 96 to 106, wherein G or G1 is independently a
    615 substituted or unsubstituted trityloxy .
    616
    617 108. The method of any one of claims 96 to 107, wherein L or L1 is independently a
    618 substituted or unsubstituted C 1 -C 12 alkylene .
    619
    620 109. The method of any one of claims 96 to 108, wherein R or R1 is independently
    621 selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    622 alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and
    623 combinations of thereof.
    624
    625 110. The method of any one of claims 96 to 109, wherein said polymer is
    626 poly(ethylene glycol).
    627
    628 111. The method of claim 110, wherein said poly(ethylene glycol) has an average
    629 molecular weight from about 500 Da to about 100,000 Da.
    630
    631 112. The method of any one of claims 96 to 111, wherein said preparation comprises at
    632 least 50% by weight of said compound.
    633
    634 113. The method of any one of claims 96 to 112, said method further comprising (i)
    635 removing the hydrophobic separation handle(s) from said structure of formula (2) and
    636 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    637 before reacting with insulin or said derivative.
    638
    639 114. The method of any one of claims 96 to 112, said method further comprising
    640 removing the hydrophobic separation handle(s) from said structure of formula (2)
    641 after reacting with insulin or said derivative.
    642
    643 115. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a structure
    644 of formula (10): B1 L3 L L L2 B
    645
    646 (10)
    647 or a salt form thereof,
    648 wherein:
    649 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    650 backbone, wherein each linking group is bonded at a different terminus of said polymer;
    651 E and E1 are independently O or S;
    652 K and Ki are independently selected from the group consisting of: alkylene,
    653 alky leneoxy alkylene, and oligomeric alkyleneoxyalkylene;
    654 G and Gi are independently absent or are selected from the group consisting of:
    655 alkoxy and a hydrophobic separation handle; 656 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    657 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    658 L and L1 are independently selected from the group consisting of: a divalent
    659 radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene,
    660 and unsubstituted and substituted arylene;
    661 L2 is a covalent linking moiety between L on the polymer backbone and B;
    662 L3 is a covalent linking moiety between L on the polymer backbone and B1; and
    663 B and B1 are independently insulin, a derivative of insulin, a biologic other than
    664 insulin, a drug, a detectable group, a separation moiety, wherein at least one of B and B1
    665 is insulin or a derivative of insulin.
    666
    667 116. A method of making an insulin conjugate, said method comprising reacting
    668 insulin or a derivative thereof with a preparation comprising a compound of formula
    669 (3):
    E
    R2 M2 polymer O P Z L M R
    670 K G (3)
    671 or a salt form thereof,
    672 wherein:
    673 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    674 backbone, wherein M2 and the phosphonate-derived functional group are bonded at a
    675 different terminus of said polymer;
    676 E and E1 are independently O or S;
    677 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    678 oligomeric alkyleneoxyalkylene;
    679 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    680 separation handle; 681 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    682 Z2 can be NH;
    683 L is selected from the group consisting of: a divalent radical of nucleoside,
    684 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    685 substituted arylene;
    686 M is selected from a protected group that when deprotected is reactive with
    687 insulin or said derivative or a group reactive with insulin or said derivative;
    688 M2 is selected from O, S or NH; and
    689 R is absent, a protecting group, a hydrophobic separation handle, or an activating
    690 group;
    691 R2 is hydrogen or a protecting group;
    692 wherein when M is a protected group that when deprotected is reactive with
    693 insulin or said derivative, then R is a protecting group or a hydrophobic separation
    694 handle; and
    695 wherein when M is a group reactive with insulin or said derivative, R is absent,
    696 hydrogen, or an activating group.
    697
    698 117. The method of claim 116, wherein one of Z1 and Z2 is NH and the other is O. 699
    700 118. The method of claim 116 or claim 117, wherein G is a substituted or
    701 unsubstituted trityloxy group.
    702
    703 119. The method of claim 117, wherein G is selected from a monoalkoxy substituted
    704 trityloxy group or dialkoxy substituted trityloxy group.
    705
    706 120. The method of any one of claims 116 to 119, wherein said group reactive with
    707 insulin or said derivative is selected from the group consisting of: hydroxyl, amine,
    708 thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    709 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    710 iodoacetamide. 71 1
    712 121. The method of any one of claims 116 to 120, wherein R2 is absent or selected 713 from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    714 alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl, 715 methyl.
    716
    717 122. The method of any one of claims 116 to 121, wherein said polymer is
    718 poly(ethylene glycol).
    719
    720 123. The method of any one of claims 1 16 to 122, wherein said preparation comprises 721 at least 50% of said compound.
    722
    723 124. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a
    724 compound of formula (11):
    726 or a salt form thereof,
    727 wherein:
    728 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer 729 backbone, wherein M2 and the phosphonate-derived functional group are bonded at a 730 different terminus of said polymer;
    731 E and E1 are independently O or S;
    732 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and 733 oligomeric alkyleneoxyalkylene;
    734 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic 735 separation handle; 736 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    737 Z2 can be NH;
    738 L is selected from the group consisting of: a divalent radical of nucleoside,
    739 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    740 substituted arylene;
    741 L2 is a covalent linking moiety between L on the polymer backbone and B;
    742 L4 is a covalent linking moiety between L on the polymer backbone and B1; and
    743 B and B1 are independently insulin, a derivative of insulin, a biologic other than
    744 insulin, a drug, a detectable group, a separation moiety, wherein at least one of B and B1
    745 is insulin or a derivative of insulin.
    746
    747 125. A method of preparing a compound comprising a water-soluble, non-peptidic, and
    748 non-nucleotidic polymer backbone having at least one terminus covalently bonded to
    749 a structure of formula (9):
    750
    E R A O P Z L L2 B
    751 Y (9)
    752 or a salt thereof,
    753 wherein:
    754 A is the point of covalent bonding to the terminus of the polymer backbone;
    755 E is O or S;
    756 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    757 heterocyclyl, aryl, and heteroaryl;
    758 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    759 Z2 can be NH; 760 L is selected from the group consisting of: a divalent radical of a nucleoside,
    761 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    762 substituted arylene;
    763 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    764 B is insulin or a derivative thereof;
    765 comprising the steps of:
    766 (a) providing a composition comprising a compound of formula (8):
    767
    769 wherein:
    770 A is the point of covalent bonding to the terminus of the polymer backbone;
    771 E is O or S;
    772 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    773 heterocyclyl, aryl, and heteroaryl;
    774 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    775 Z2 can be NH;
    776 L is selected from the group consisting of: a divalent radical of a nucleoside,
    777 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    778 substituted arylene;
    779 M is a protected group that when deprotected is reactive with insulin or a
    780 derivative thereof;
    781 R is a hydrophobic separation handle;
    782 R1 is absent or a hydrophobic separation handle; and
    783 (b) removing the hydrophobic separation handle(s);
    784 (c) optionally reacting the compound obtained in step (b) with an activating agent;
    785 and 786 (d) reacting the compound obtained in step (b), or, optionally in step (c), with
    787 insulin or a derivative thereof.
    788
    789 126. The method of claim 125, wherein Z1 is O and Z2 is NH.
    790
    791 127. The method of claim 125, wherein Z1 is NH and Z2 is O.
    792
    793 128. The method of claim 125, wherein both Z1 and Z2 are O.
    794
    795 129. The method of any one of claims 125 to 128, wherein the protected group M,
    796 when deprotected, is represented by hydroxyl, amine, thiol, carboxyl, aldehyde,
    797 glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,
    798 hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide.
    799
    800 130. The method of any one of claims 125 to 129, wherein R is selected from the
    801 group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    802 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    803
    804 131. The method of any one of claims 125 to 130, wherein said polymer backbone is
    805 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    806 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    807 polyoxazoline, and copolymers.
    808
    809 132. The method of any one of claims 125 to 131, wherein said polymer backbone is
    810 poly(ethylene glycol).
    81 1
    812 133. The method of any one of claims 125 to 132, wherein reaction step (d) is carried
    813 out in the presence of water or a protic solvent.
    814
    815 134. The method of any one of claims 125 to 133, wherein the compound of formula
    816 (8) comprises at least 98% by weight of said composition.
    817
    818 135. A composition comprising the conjugate of any one of claims 95, 115, and 124,
    819 and a pharmaceutically acceptable excipient.
    820
    821 136. The composition of claim 135, wherein the polymer backbone of said conjugate is
    822 poly(ethylene glycol).
    823
    824 137. The composition of claim 135 or claim 136, wherein B is insulin.
    825
    826 138. The composition of any one of claims 135 to 137, wherein B1 is insulin.
    827
    828 139. A method of treating a patient diagnosed with diabetes, said method comprising
    829 administering to said patient an effective amount of the conjugate of any one of
    830 claims 95, 115, or 124.
    831
    832 140. The method of claim 139, wherein the polymer backbone of said conjugate is
    833 poly(ethylene glycol).
    834
    835 141. The method of claim 139 or claim 140, wherein B is insulin.
    836
    837 142. The method of any one of claims 139 to 141, wherein B1 is insulin.
    838
    839 143. A method of making an Omalizumab conjugate, said method comprising reacting
    840 Omalizumab or a derivative thereof with a preparation comprising a water-soluble,
    841 non-peptidic, and non-nucleotidic polymer backbone having at least one terminus
    842 covalently bonded to a structure of formula (1) under conditions suitable for group M
    843 or R to react with Omalizumab or said derivative thereof:
    844
    846 or a salt thereof,
    847 wherein:
    848 A is the point of covalent bonding to the terminus of the polymer backbone;
    849 E is O or S;
    850 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    851 oligomeric alkyleneoxyalkylene;
    852 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    853 separation handle;
    854 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    855 can be NH;
    856 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    857 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    858 substituted arylene;
    859 M is a protected group that when deprotected is reactive with Omalizumab or
    860 derivative thereof or is a group reactive with Omalizumab or derivative thereof;
    861 R is absent or selected from the group consisting of: hydrogen, a protecting group, a
    862 hydrophobic separation handle, or an activating group;
    863 R1 is absent or a hydrophobic separation handle;
    864 wherein when M is a protected group that when deprotected is reactive with
    865 Omalizumab or derivative thereof, then R is a protecting group or a hydrophobic
    866 separation handle;
    867 wherein when M is a group reactive with Omalizumab or derivative thereof, R is
    868 absent, hydrogen, or an activating group; and
    869 wherein only one of R, R1, and G can be a hydrophobic separation handle, . 870
    871 144. The method of claim 143, wherein the polymer has from 2 to 100 termini.
    872
    873 145. The method of claim 143 or claim 144, wherein only one termini of the polymer
    874 backbone is covalently bonded to the structure of formula (1).
    875
    876 146. The method of any one of claims 143 to 145 wherein the polymer backbone has
    877 two termini.
    878
    879 147. The method of claim 146, wherein only one termini of the polymer backbone is
    880 covalently bonded to the structure of formula (1).
    881
    882 148. The method of claim 146, wherein both termini of the polymer backbone are
    883 covalently bonded to the structure of formula (1).
    884
    885 149. The method of any one of claims 143 to 148, wherein one of Z1 and Z2 is NH and
    886 the other is O.
    887
    888 150. The method of claim 149, wherein Z1 is O and Z2 is NH.
    889
    890 151. The method of claim 149, wherein Z1 is NH and Z2 is O.
    891
    892 152. The method of claim 149, wherein both Z1 and Z2 are O.
    893
    894 153. The method of any one of claims 143 to 152, wherein said group reactive with
    895 Omalizumab or said derivative is selected from the group consisting of: hydroxyl,
    896 amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    897 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    898 iodoacetamide.
    899
    900 154. The method of any one of claims 143 to 153, wherein K is selected from the
    901 group consisting of: methylene, ethylene, propylene, isopropylene, butylene,
    902 isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene
    903 glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    904
    905 155. The method of any one of claims 143 to 154 wherein G is a substituted or
    906 unsubstituted trityloxy.
    907
    908 156. The method of any one of claims 143 to 155, wherein L is a substituted or
    909 unsubstituted alkylene.
    910
    91 1 157. The method any one of claims 143 to 156, wherein R is selected from the group
    912 consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    913 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    914
    915 158. The method of any one of claims 143 to 157, wherein said group reactive with
    916 Omalizumab or said derivative is carboxyl and R is absent or selected from the group
    917 consisting of: N-hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl.
    918
    919 159. The method of any one of claims 143 to 158, wherein said polymer backbone is
    920 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    921 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    922 polyoxazoline, and copolymers.
    923
    924 160. The method of any one of claims 143 to 159, wherein said polymer backbone is
    925 poly(ethylene glycol).
    926
    927 161. The method of claim 160, wherein said poly(ethylene glycol) has an average
    928 molecular weight from about 500 Da to about 100,000 Da.
    929
    930 162. The method of any one of claims 143 to 161, wherein said preparation comprises
    931 at least 50% by weight of said water-soluble, non-peptidic, and non-nucleotidic
    932 polymer backbone having at least one terminus covalently bonded to a structure of
    933 formula (1).
    934
    935 163. The method of claim 162, wherein said preparation comprises at least 98% by
    936 weight of said water-soluble, non-peptidic, and non-nucleotidic polymer backbone
    937 having at least one terminus covalently bonded to a structure of formula (1).
    938
    939 164. The method of any one of claims 143 to 163, said method further comprising (i)
    940 removing the hydrophobic separation handle(s) from said structure of formula (1) and
    941 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    942 before reacting with Omalizumab or said derivative.
    943
    944 165. The method of any one of claims 143 to 163, said method further comprising
    945 removing the hydrophobic separation handle(s) from said structure of formula (1)
    946 after reacting with Omalizumab or said derivative.
    947
    948 166. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a water-
    949 soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one
    950 terminus covalently bonded to a structure of formula (9):
    R
    A O 1 L L2 B
    951
    952 or a salt thereof,
    953 wherein:
    954 A is the point of covalent bonding to the terminus of the polymer backbone; 955 E is O or S;
    956 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    957 oligomeric alkyleneoxyalkylene;
    958 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    959 separation handle;
    960 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    961 can be NH;
    962 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    963 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    964 substituted arylene;
    965 R1 is absent or a hydrophobic separation handle, wherein only one of R1 and G can be
    966 a hydrophobic separation handle;
    967 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    968 B is Omalizumab or a derivative thereof.
    969
    970 167. A method of making an Omalizumab conjugate, said method comprising reacting
    971 Omalizumab or a derivative thereof with a preparation comprising a compound of
    972 formula (2) under conditions suitable for group M or R to react with Omalizumab or
    973 said derivative thereof:
    E1 E
    R1 1 -Z3 P O polymer- -O- -M R
    \
    974 (2) 975 or a salt form thereof,
    976 wherein: 977 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    978 backbone, wherein each linking group is bonded at a different terminus of said
    979 polymer;
    980 E and E1 are independently O or S;
    981 K and Ki are independently selected from the group consisting of: alkylene,
    982 alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    983 G and Gi are independently absent or are selected from the group consisting of:
    984 alkoxy and a hydrophobic separation handle;
    985 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    986 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    987 L and L1 are independently selected from the group consisting of: a divalent radical of
    988 a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and
    989 unsubstituted and substituted arylene;
    990 M and M1 are independently selected from a protected group that when deprotected is
    991 reactive with Omalizumab or said derivative or a group reactive with
    992 Omalizumab or said derivative, wherein M and M1 are different; and
    993 R and R1 are independently absent, hydrogen, a protecting group, or an activating
    994 group;
    995 wherein when M is a protected group that when deprotected is reactive with
    996 Omalizumab or said derivative, then R is a protecting group or a hydrophobic
    997 separation handle;
    998 wherein when M is a group reactive with Omalizumab or said derivative, R is absent,
    999 hydrogen, or an activating group;
    000 wherein when M1 is a protected group that when deprotected is reactive with
    001 Omalizumab or said derivative, then R1 is a protecting group or a hydrophobic
    002 separation handle; and
    003 wherein when M1 is a group reactive with Omalizumab or said derivative, R1 is
    004 absent, hydrogen, or an activating group.
    005
    006 168. The method of claim 167, wherein one of Z1 and Z2 is NH and the other is O.
    169. The method of claim 168, wherein Z1 is O and Z2 is NH. 170. The method of claim 168, wherein Z1 is NH and Z2 is O. 171. The method of claim 167, wherein Z1 is O and Z2 is O. 172. The method of claim 167, wherein one of Z3 and Z4 is NH and the other is O. 173. The method of claim 172, wherein Z3 is O and Z4 is NH. 174. The method of claim 172, wherein Z3 is NH and Z4 is O. 175. The method of claim 167, wherein Z3 is O and Z4 is O. 176. The method of any one of claims 167 to 175, wherein said group reactive with Omalizumab or said derivative thereof is selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide. 177. The method of any one of claims 167 to 176, wherein K or K1 is independently selected from the group consisting of: methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 178. The method of any one of claims 167 to 177, wherein G or G1 is independently a substituted or unsubstituted trityloxy.
    179. The method of any one of claims 167 to 178, wherein L or L1 is independently a substituted or unsubstituted alkylene. 180. The method of any one of claims 167 to 179, wherein R or R1 is independently selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and combinations of thereof. 181. The method of any one of claims 167 to 180, wherein said polymer is
    poly(ethylene glycol). 182. The method of claim 181, wherein said poly(ethylene glycol) has an average molecular weight from about 500 Da to about 100,000 Da. 183. The method of any one of claims 167 to 182, wherein said preparation comprises at least 50% by weight of said compound. 184. The method of any one of claims 167 to 183, said method further comprising (i) removing the hydrophobic separation handle(s) from said structure of formula (2) and (ii) optionally reacting the compound obtained in step (i) with an activating agent before reacting with Omalizumab or said derivative. 185. The method of any one of claims 167 to 183, said method further comprising removing the hydrophobic separation handle(s) from said structure of formula (2) after reacting with Omalizumab or said derivative. 186. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a structure of formula (10): L3 L1 Z3 P O polymer O P Z1 L L2 B
    (10) or a salt form thereof,
    wherein:
    polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer backbone, wherein each linking group is bonded at a different terminus of said polymer;
    E and E1 are independently O or S;
    K and Ki are independently selected from the group consisting of:
    alkylene, alkyleneoxyalkylene, and oligomeric alky leneoxy alky lene;
    G and Gi are independently absent or are selected from the group consisting of: alkoxy and a hydrophobic separation handle;
    each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH, wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    L and L1 are independently selected from the group consisting of: a divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and substituted arylene;
    L2 is a covalent linking moiety between L on the polymer backbone and B;
    L3 is a covalent linking moiety between L on the polymer backbone and B1; and
    B and B1 are independently Omalizumab, a derivative of Omalizumab, a biologic other than Omalizumab, a drug, a detectable group, a separation moiety, wherein at least one of B and B1 is Omalizumab or a derivative of Omalizumab.
    088 187. A method of making an Omalizumab conjugate, said method comprising reacting
    089 Omalizumab or a derivative thereof with a preparation comprising a compound of
    090 formula (3):
    E
    R2 M2 polymer O P Z L M R
    091 K G (3)
    092 or a salt form thereof,
    093 wherein:
    094 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    095 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    096 at a different terminus of said polymer;
    097 E and E1 are independently O or S;
    098 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    099 oligomeric alkyleneoxyalkylene;
    100 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic 1 o 1 separation handle;
    102 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    103 can be NH;
    104 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    105 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    106 substituted arylene;
    107 M is selected from a protected group that when deprotected is reactive with
    108 Omalizumab or said derivative or a group reactive with Omalizumab or said
    109 derivative;
    1 10 M2 is selected from O, S or NH; and
    1 1 1 R is absent, a protecting group, a hydrophobic separation handle, or an activating
    1 12 group;
    1 13 R2 is hydrogen or a protecting group; wherein when M is a protected group that when deprotected is reactive with
    Omalizumab or said derivative, then R is a protecting group or a hydrophobic separation handle; and
    wherein when M is a group reactive with Omalizumab or said derivative, R is absent, hydrogen, or an activating group. 188. The method of claim 187, wherein one of Z1 and Z2 is NH and the other is O. 189. The method of claim 187 or claim 188, wherein G is a substituted or
    unsubstituted trityloxy group . 190. The method of claim 187, wherein G is selected from a monoalkoxy substituted trityloxy group or dialkoxy substituted trityloxy group. 191. The method of any one of claims 187 to 190, wherein said group reactive with Omalizumab or said derivative is selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide.
    192. The method of any one of claims 187 to 191, wherein R2 is absent or selected from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl, methyl. 193. The method of any one of claims 187 to 192, wherein said polymer is
    poly(ethylene glycol). 194. The method of any one of claims 187 to 193, wherein said preparation comprises at least 50% of said compound.
    195. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a
    compound of formula (11):
    146
    147 or a salt form thereof,
    148 wherein:
    149 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    150 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    151 at a different terminus of said polymer;
    152 E and E1 are independently O or S;
    153 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    154 oligomeric alkyleneoxyalkylene;
    155 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    156 separation handle;
    157 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    158 can be NH;
    159 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    160 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    161 substituted arylene;
    162 L2 is a covalent linking moiety between L on the polymer backbone and B;
    163 L4 is a covalent linking moiety between L on the polymer backbone and B1; and
    164 B and B1 are independently Omalizumab, a derivative of Omalizumab, a biologic
    165 other than Omalizumab, a drug, a detectable group, a separation moiety, wherein
    166 at least one of B and B1 is Omalizumab or a derivative of Omalizumab.
    167
    168 196. A method of preparing a compound comprising a water-soluble, non-peptidic, and
    169 non-nucleotidic polymer backbone having at least one terminus covalently bonded to
    170 a structure of formula (9):
    171
    E R A O P Z L L2 B
    172 Y (9)
    173 or a salt thereof,
    174 wherein:
    175 A is the point of covalent bonding to the terminus of the polymer backbone;
    176 E is O or S;
    177 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    178 heterocyclyl, aryl, and heteroaryl;
    179 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    180 can be NH;
    181 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    182 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    183 substituted arylene;
    184 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    185 B is Omalizumab or a derivative thereof;
    186 comprising the steps of:
    187 (a) providing a composition comprising a compound of formula (8):
    188
    190 wherein: 191 A is the point of covalent bonding to the terminus of the polymer backbone;
    192 E is O or S;
    193 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    194 heterocyclyl, aryl, and heteroaryl;
    195 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    196 can be NH;
    197 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    198 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    199 substituted arylene;
    200 M is a protected group that when deprotected is reactive with Omalizumab or a
    201 derivative thereof;
    202 R is a hydrophobic separation handle;
    203 R1 is absent or a hydrophobic separation handle; and
    204 (b) removing the hydrophobic separation handle(s);
    205 (c) optionally reacting the compound obtained in step (b) with an activating agent;
    206 and
    207 (d) reacting the compound obtained in step (b), or, optionally in step (c), with
    208 Omalizumab or a derivative thereof.
    209
    210 197. The method of claim 196, wherein Z1 is O and Z2 is NH.
    21 1
    212 198. The method of claim 196, wherein Z1 is NH and Z2 is O.
    213
    214 199. The method of claim 196, wherein both Z1 and Z2 are O.
    215
    216 200. The method of any one of claims 196 to 199, wherein the protected group M,
    217 when deprotected, is represented by hydroxyl, amine, thiol, carboxyl, aldehyde,
    218 glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,
    219 hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide.
    220
    221 201. The method of any one of claims 196 to 200, wherein R is selected from the
    222 group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    223 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    224
    225 202. The method of any one of claims 196 to 201, wherein said polymer backbone is
    226 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    227 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    228 polyoxazoline, and copolymers.
    229
    230 203. The method of any one of claims 196 to 202, wherein said polymer backbone is
    231 poly(ethylene glycol).
    232
    233 204. The method of any one of claims 196 to 203, wherein reaction step (d) is carried
    234 out in the presence of water or a protic solvent.
    235
    236 205. The method of any one of claims 196 to 204, wherein the compound of formula
    237 (8) comprises at least 98% by weight of said composition.
    238
    239 206. A composition comprising the conjugate of any one of claims 167, 187, and 196,
    240 and a pharmaceutically acceptable excipient.
    241
    242 207. The composition of claim 206, wherein the polymer backbone of said conjugate is
    243 poly(ethylene glycol).
    244
    245 208. The composition of claim 206 or claim 207, wherein B is Omalizumab.
    246
    247 209. The composition of any one of claims 206 to 207, wherein B1 is Omalizumab. 248
    249 210. A method of treating a patient diagnosed with asthma, said method comprising
    250 administering to said patient an effective amount of the conjugate of any one of
    251 claims 167, 187, and 196.
    252
    253 211. The method of claim 210, wherein the polymer backbone of said conjugate is
    254 poly(ethylene glycol).
    255
    256 212. The method of claim 210 or claim 211, wherein B is Omalizumab .
    257
    258 213. The method of any one of claims 210 to 211 , wherein B 1 is Omalizumab .
    259
    260 214. A method of making an a clotting factor conjugate, said method comprising
    261 reacting a clotting factor or a derivative thereof with a preparation comprising a
    262 water-soluble, non-peptidic, and non-nucleotidic polymer backbone having at least
    263 one terminus covalently bonded to a structure of formula (1) under conditions suitable
    264 for group M or R to react with a clotting factor or said derivative thereof:
    265
    267 or a salt thereof,
    268 wherein:
    269 A is the point of covalent bonding to the terminus of the polymer backbone;
    270 E is O or S;
    271 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    272 oligomeric alkyleneoxyalkylene; 273 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    274 separation handle;
    275 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    276 can be NH;
    277 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    278 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    279 substituted arylene;
    280 M is a protected group that when deprotected is reactive with a clotting factor or
    281 derivative thereof or is a group reactive with a clotting factor or derivative
    282 thereof;
    283 R is absent or selected from the group consisting of: hydrogen, a protecting group, a
    284 hydrophobic separation handle, or an activating group;
    285 R1 is absent or a hydrophobic separation handle;
    286 wherein when M is a protected group that when deprotected is reactive with a a
    287 clotting factor or derivative thereof, then R is a protecting group or a hydrophobic
    288 separation handle;
    289 wherein when M is a group reactive with a clotting factor or derivative thereof, R is
    290 absent, hydrogen, or an activating group; and
    291 wherein only one of R, R1, and G can be a hydrophobic separation handle, .
    292
    293 215. The method of claim 214, wherein the polymer has from 2 to 100 termini.
    294
    295 216. The method of claim 214 or claim 215, wherein only one termini of the polymer
    296 backbone is covalently bonded to the structure of formula (1).
    297
    298 217. The method of any one of claims 214 to 216 wherein the polymer backbone has
    299 two termini.
    300
    301 218. The method of claim 217, wherein only one termini of the polymer backbone is
    302 covalently bonded to the structure of formula (1). 303
    304 219. The method of claim 217, wherein both termini of the polymer backbone are
    305 covalently bonded to the structure of formula (1).
    306
    307 220. The method of any one of claims 214 to 219, wherein one of Z1 and Z2 is NH and
    308 the other is O.
    309
    310 221. The method of claim 210, wherein Z1 is O and Z2 is NH.
    31 1
    312 222. The method of claim 210, wherein Z1 is NH and Z2 is O.
    313
    314 223. The method of claim 210, wherein both Z1 and Z2 are O.
    315
    316 224. The method of any one of claims 214 to 223, wherein said group reactive with a
    317 clotting factor or said derivative is selected from the group consisting of: hydroxyl,
    318 amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    319 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    320 iodoacetamide.
    321
    322 225. The method of any one of claims 214 to 224, wherein K is selected from the
    323 group consisting of: methylene, ethylene, propylene, isopropylene, butylene,
    324 isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene
    325 glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    326
    327 226. The method of any one of claims 214 to 225, wherein G is a substituted or
    328 unsubstituted trityloxy.
    329
    330 227. The method of any one of claims 214 to 226, wherein L is a substituted or
    331 unsubstituted alkylene.
    332
    333 228. The method any one of claims 214 to 227, wherein R is selected from the group
    334 consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    335 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    336
    337 229. The method of any one of claims 214 to 228, wherein said group reactive with a
    338 clotting factor or said derivative is carboxyl and R is absent or selected from the
    339 group consisting of: N-hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl. 340
    341 230. The method of any one of claims 214 to 229, wherein said polymer backbone is
    342 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    343 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    344 polyoxazoline, and copolymers.
    345
    346 231. The method of any one of claims 214 to 230, wherein said polymer backbone is
    347 poly(ethylene glycol).
    348
    349 232. The method of claim 231 , wherein said poly(ethylene glycol) has an average
    350 molecular weight from about 500 Da to about 100,000 Da.
    351
    352 233. The method of any one of claims 214 to 232, wherein said preparation comprises
    353 at least 50% by weight of said water-soluble, non-peptidic, and non-nucleotidic
    354 polymer backbone having at least one terminus covalently bonded to a structure of
    355 formula (1).
    356
    357 234. The method of claim 233, wherein said preparation comprises at least 98% by
    358 weight of said water-soluble, non-peptidic, and non-nucleotidic polymer backbone
    359 having at least one terminus covalently bonded to a structure of formula (1).
    360
    361 235. The method of any one of claims 214 to 234, said method further comprising (i)
    362 removing the hydrophobic separation handle(s) from said structure of formula (1) and 363 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    364 before reacting with a clotting factor or said derivative.
    365
    366 236. The method of any one of claims 214 to 235, said method further comprising
    367 removing the hydrophobic separation handle(s) from said structure of formula (1)
    368 after reacting with a clotting factor or said derivative.
    369
    370 237. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a water-
    371 soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one
    372 terminus covalently bonded to a structure of formula (9):
    R
    A O 1 L L2 B
    373
    374 or a salt thereof,
    375 wherein:
    376 A is the point of covalent bonding to the terminus of the polymer backbone;
    377 E is O or S;
    378 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    379 oligomeric alkyleneoxyalkylene;
    380 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    381 separation handle;
    382 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    383 can be NH;
    384 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    385 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    386 substituted arylene; 387 R1 is absent or a hydrophobic separation handle, wherein only one of R1 and G can be
    388 a hydrophobic separation handle;
    389 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    390 B is a clotting factor or a derivative thereof.
    391
    392 238. A method of making a clotting factor conjugate, said method comprising reacting
    393 a clotting factor or a derivative thereof with a preparation comprising a compound of
    394 formula (2) under conditions suitable for group M or R to react with a clotting factor
    395 or said derivative thereof:
    E1 E
    R1 1 -Z3 P O polymer- -O- -M R
    / \
    396 (2)
    397 or a salt form thereof,
    398 wherein:
    399 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    400 backbone, wherein each linking group is bonded at a different terminus of said
    401 polymer;
    402 E and E1 are independently O or S;
    403 K and Ki are independently selected from the group consisting of: alkylene,
    404 alky leneoxy alkylene, and oligomeric alkyleneoxyalkylene;
    405 G and Gi are independently absent or are selected from the group consisting of:
    406 alkoxy and a hydrophobic separation handle;
    407 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    408 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH; 409 L and L1 are independently selected from the group consisting of: a divalent radical of
    410 a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and
    41 1 unsubstituted and substituted arylene;
    412 M and M1 are independently selected from a protected group that when deprotected is
    413 reactive with a clotting factor or said derivative or a group reactive with a clotting
    414 factor or said derivative, wherein M and M1 are different; and
    415 R and R1 are independently absent, hydrogen, a protecting group, or an activating
    416 group;
    417 wherein when M is a protected group that when deprotected is reactive with a clotting
    418 factor or said derivative, then R is a protecting group or a hydrophobic separation
    419 handle;
    420 wherein when M is a group reactive with a clotting factor or said derivative, R is
    421 absent, hydrogen, or an activating group;
    422 wherein when M1 is a protected group that when deprotected is reactive with a
    423 clotting factor or said derivative, then R1 is a protecting group or a hydrophobic
    424 separation handle; and
    425 wherein when M1 is a group reactive with a clotting factor or said derivative, R1 is
    426 absent, hydrogen, or an activating group.
    427
    428 239. The method of claim 238, wherein one of Z1 and Z2 is NH and the other is O. 429
    430 240. The method of claim 239, wherein Z1 is O and Z2 is NH.
    431
    432 241. The method of claim 239, wherein Z1 is NH and Z2 is O.
    433
    434 242. The method of claim 238, wherein Z1 is O and Z2 is O.
    435
    436 243. The method of claim 238, wherein one of Z3 and Z4 is NH and the other is O. 437
    438 244. The method of claim 243, wherein Z3 is O and Z4 is NH. 439
    440 245. The method of claim 243, wherein Z3 is NH and Z4 is O.
    441
    442 246. The method of claim 238, wherein Z3 is O and Z4 is O.
    443
    444 247. The method of any one of claims 238 to 246, wherein said group reactive with a
    445 clotting factor or said derivative thereof is selected from the group consisting of:
    446 hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl,
    447 alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,
    448 dithiopyridine, iodoacetamide.
    449
    450 248. The method of any one of claims 238 to 247, wherein K or K1 is independently
    451 selected from the group consisting of: methylene, ethylene, propylene, isopropylene,
    452 butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from
    453 diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 454
    455 249. The method of any one of claims 238 to 248, wherein G or G1 is independently a
    456 substituted or unsubstituted trityloxy.
    457
    458 250. The method of any one of claims 238 to 249, wherein L or L1 is independently a
    459 substituted or unsubstituted alkylene.
    460
    461 251. The method of any one of claims 238 to 250, wherein R or R1 is independently
    462 selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    463 alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and
    464 combinations of thereof.
    465
    466 252. The method of any one of claims 238 to 251 , wherein said polymer is
    467 poly(ethylene glycol).
    468
    469 253. The method of claim 252, wherein said poly(ethylene glycol) has an average
    470 molecular weight from about 500 Da to about 100,000 Da.
    471
    472 254. The method of any one of claims 238 to 253, wherein said preparation comprises
    473 at least 50% by weight of said compound.
    474
    475 255. The method of any one of claims 238 to 254, said method further comprising (i)
    476 removing the hydrophobic separation handle(s) from said structure of formula (2) and
    477 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    478 before reacting with a clotting factor or said derivative.
    479
    480 256. The method of any one of claims 238 to 254, said method further comprising
    481 removing the hydrophobic separation handle(s) from said structure of formula (2)
    482 after reacting with a clotting factor or said derivative.
    483
    484 257. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a structure
    485 of formula (10):
    E1 E B1 L3 L1 Z3 P O polymer O P Z1 L L2 B
    Z4 Z2
    G 11 K /1 \ K G
    486 (10)
    487 or a salt form thereof,
    488 wherein:
    489 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic
    490 polymer backbone, wherein each linking group is bonded at a different terminus
    491 of said polymer;
    492 E and E1 are independently O or S; 493 K and Ki are independently selected from the group consisting of:
    494 alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    495 G and Gi are independently absent or are selected from the group
    496 consisting of: alkoxy and a hydrophobic separation handle;
    497 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O
    498 and NH, wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    499 L and L1 are independently selected from the group consisting of: a
    500 divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric
    501 alkyleneoxyalkylene, and unsubstituted and substituted arylene;
    502 L2 is a covalent linking moiety between L on the polymer backbone and
    503 B;
    504 L3 is a covalent linking moiety between L on the polymer backbone and
    505 B1; and
    506 B and B1 are independently a clotting factor, a derivative of a clotting
    507 factor, a biologic other than a clotting factor, a drug, a detectable group, a
    508 separation moiety, wherein at least one of B and B1 is a clotting factor or a
    509 derivative of a clotting factor.
    510
    51 1 258. A method of making a clotting factor conjugate, said method comprising reacting
    512 a clotting factor or a derivative thereof with a preparation comprising a compound of
    513 formula (3):
    E
    R2 M2 polymer O P Z L M R
    514 K G (3)
    515 or a salt form thereof,
    516 wherein: 517 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    518 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    519 at a different terminus of said polymer;
    520 E and E1 are independently O or S;
    521 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    522 oligomeric alkyleneoxyalkylene;
    523 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    524 separation handle;
    525 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    526 can be NH;
    527 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    528 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    529 substituted arylene;
    530 M is selected from a protected group that when deprotected is reactive with a clotting
    531 factor or said derivative or a group reactive with a clotting factor or said
    532 derivative;
    533 M2 is selected from O, S or NH; and
    534 R is absent, a protecting group, a hydrophobic separation handle, or an activating
    535 group;
    536 R2 is hydrogen or a protecting group;
    537 wherein when M is a protected group that when deprotected is reactive with a clotting
    538 factor or said derivative, then R is a protecting group or a hydrophobic separation
    539 handle; and
    540 wherein when M is a group reactive with a clotting factor or said derivative, R is
    541 absent, hydrogen, or an activating group.
    542
    543 259. The method of claim 258, wherein one of Z1 and Z2 is NH and the other is O. 544
    545 260. The method of claim 258 or claim 259, wherein G is a substituted or
    546 unsubstituted trityloxy group.
    261. The method of claim 258, wherein G is selected from a monoalkoxy substituted trityloxy group or dialkoxy substituted trityloxy group.
    262. The method of any one of claims 258 to 261, wherein said group reactive with a clotting factor or said derivative is selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide.
    263. The method of any one of claims 258 to 262, wherein R2 is absent or selected from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl, methyl.
    264. The method of any one of claims 258 to 263, wherein said polymer is
    poly(ethylene glycol).
    265. The method of any one of claims 258 to 264, wherein said preparation comprises at least 50% of said compound.
    266. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a
    compound of formula (11):
    or a salt form thereof,
    wherein: 572 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    573 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    574 at a different terminus of said polymer;
    575 E and E1 are independently O or S;
    576 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    577 oligomeric alkyleneoxyalkylene;
    578 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    579 separation handle;
    580 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    581 can be NH;
    582 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    583 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    584 substituted arylene;
    585 L2 is a covalent linking moiety between L on the polymer backbone and B;
    586 L4 is a covalent linking moiety between L on the polymer backbone and B1; and
    587 B and B1 are independently a clotting factor, a derivative of a clotting factor, a
    588 biologic other than a clotting factor, a drug, a detectable group, a separation
    589 moiety, wherein at least one of B and B1 is a clotting factor or a derivative of a
    590 clotting factor.
    591
    592 267. A method of preparing a compound comprising a water-soluble, non-peptidic, and
    593 non-nucleotidic polymer backbone having at least one terminus covalently bonded to
    594 a structure of formula (9):
    595
    597 or a salt thereof, 598 wherein:
    599 A is the point of covalent bonding to the terminus of the polymer backbone;
    600 E is O or S;
    601 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    602 heterocyclyl, aryl, and heteroaryl;
    603 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    604 can be NH;
    605 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    606 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    607 substituted arylene;
    608 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    609 B is a clotting factor or a derivative thereof;
    610 comprising the steps of:
    61 1 (a) providing a composition comprising a compound of formula (8):
    612
    614 wherein:
    615 A is the point of covalent bonding to the terminus of the polymer backbone;
    616 E is O or S;
    617 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    618 heterocyclyl, aryl, and heteroaryl;
    619 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    620 can be NH;
    621 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    622 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    623 substituted arylene; 624 M is a protected group that when deprotected is reactive with a clotting factor or a
    625 derivative thereof;
    626 R is a hydrophobic separation handle;
    627 R1 is absent or a hydrophobic separation handle; and
    628 (b) removing the hydrophobic separation handle(s);
    629 (c) optionally reacting the compound obtained in step (b) with an activating agent;
    630 and
    631 (d) reacting the compound obtained in step (b), or, optionally in step (c), with a
    632 clotting factor or a derivative thereof.
    633
    634 268. The method of claim 267, wherein Z1 is O and Z2 is NH.
    635
    636 269. The method of claim 267, wherein Z1 is NH and Z2 is O.
    637
    638 270. The method of claim 267, wherein both Z1 and Z2 are O.
    639
    640 271. The method of any one of claims 267 to 270, wherein the protected group M,
    641 when deprotected, is represented by hydroxyl, amine, thiol, carboxyl, aldehyde,
    642 glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,
    643 hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide.
    644
    645 272. The method of any one of claims 267 to 271, wherein R is selected from the
    646 group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    647 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    648
    649 273. The method of any one of claims 267 to 272, wherein said polymer backbone is
    650 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    651 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    652 polyoxazoline, and copolymers.
    653
    654 274. The method of any one of claims 267 to 273, wherein said polymer backbone is
    655 poly(ethylene glycol).
    656
    657 275. The method of any one of claims 267 to 274, wherein reaction step (d) is carried
    658 out in the presence of water or a protic solvent.
    659
    660 276. The method of any one of claims 267 to 275, wherein the compound of formula
    661 (8) comprises at least 98% by weight of said composition.
    662
    663 277. A composition comprising the conjugate of any one of claims 238, 258, and 267,
    664 and a pharmaceutically acceptable excipient.
    665
    666 278. The composition of claim 277, wherein the polymer backbone of said conjugate is
    667 poly(ethylene glycol).
    668
    669 279. The composition of claim 277 or claim 278, wherein B is a clotting factor.
    670
    671 280. The composition of any one of claims 277 to 279, wherein B1 is a clotting factor. 672
    673 281. A method of treating a patient having hemophilia, said method comprising
    674 administering to said patient an effective amount of the conjugate of any one of
    675 claims 238, 258, or 267.
    676
    677 282. The method of claim 281 , wherein the polymer backbone of said conjugate is
    678 poly(ethylene glycol).
    679
    680 283. The method of claim 281 or claim 282, wherein B is a clotting factor.
    681
    682 284. The method of any one of claims 281 to 283, wherein B1 is a clotting factor. 683
    684 285. A method of making a polypeptide that boosts red or white cell production
    685 conjugate, said method comprising reacting a polypeptide that boosts red or white cell
    686 production or a derivative thereof with a preparation comprising a water-soluble, non- 687 peptidic, and non-nucleotidic polymer backbone having at least one terminus
    688 covalently bonded to a structure of formula (1) under conditions suitable for group M
    689 or R to react with a polypeptide that boosts red or white cell production or said
    690 derivative thereof:
    691
    693 or a salt thereof,
    694 wherein:
    695 A is the point of covalent bonding to the terminus of the polymer backbone;
    696 E is O or S;
    697 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    698 oligomeric alkyleneoxyalkylene;
    699 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    700 separation handle;
    701 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    702 can be NH;
    703 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    704 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    705 substituted arylene;
    706 M is a protected group that when deprotected is reactive with a polypeptide that
    707 boosts red or white cell production or derivative thereof or is a group reactive
    708 with a polypeptide that boosts red or white cell production or derivative thereof; 709 R is absent or selected from the group consisting of: hydrogen, a protecting group, a
    710 hydrophobic separation handle, or an activating group;
    71 1 R1 is absent or a hydrophobic separation handle;
    712 wherein when M is a protected group that when deprotected is reactive with a a
    713 polypeptide that boosts red or white cell production or derivative thereof, then R
    714 is a protecting group or a hydrophobic separation handle;
    715 wherein when M is a group reactive with a polypeptide that boosts red or white cell
    716 production or derivative thereof, R is absent, hydrogen, or an activating group;
    717 and
    718 wherein only one of R, R1, and G can be a hydrophobic separation handle, .
    719
    720 286. The method of claim 285, wherein the polymer has from 2 to 100 termini.
    721
    722 287. The method of claim 285 or claim 286, wherein only one termini of the polymer
    723 backbone is covalently bonded to the structure of formula (1).
    724
    725 288. The method of any one of claims 285 to 257 wherein the polymer backbone has
    726 two termini.
    727
    728 289. The method of claim 288, wherein only one termini of the polymer backbone is
    729 covalently bonded to the structure of formula (1).
    730
    731 290. The method of claim 288, wherein both termini of the polymer backbone are
    732 covalently bonded to the structure of formula (1).
    733
    734 291. The method of any one of claims 285 to 290, wherein one of Z1 and Z2 is NH and
    735 the other is O.
    736
    737 292. The method of claim 291 , wherein Z1 is O and Z2 is NH.
    738
    739 293. The method of claim 291 , wherein Z1 is NH and Z2 is O.
    740
    741 294. The method of claim 291 , wherein both Z1 and Z2 are O.
    742
    743 295. The method of any one of claims 285 to 294, wherein said group reactive with a
    744 polypeptide that boosts red or white cell production or said derivative is selected from
    745 the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione,
    746 alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy,
    747 maleimide, dithiopyridine, iodoacetamide.
    748
    749 296. The method of any one of claims 285 to 295, wherein K is selected from the
    750 group consisting of: methylene, ethylene, propylene, isopropylene, butylene,
    751 isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene
    752 glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    753
    754 297. The method of any one of claims 285 to 296, wherein G is a substituted or
    755 unsubstituted trityloxy.
    756
    757 298. The method of any one of claims 285 to 297, wherein L is a substituted or
    758 unsubstituted alkylene.
    759
    760 299. The method any one of claims 285 to 298, wherein R is selected from the group
    761 consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    762 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    763
    764 300. The method of any one of claims 285 to 299, wherein said group reactive with a
    765 polypeptide that boosts red or white cell production or said derivative is carboxyl and
    766 R is absent or selected from the group consisting of: N-hydroxysuccinimidyl, p-
    767 nitrophenyl, or pentachlorophenyl.
    768 301. The method of any one of claims 285 to 300, wherein said polymer backbone is
    769 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    770 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    771 polyoxazoline, and copolymers.
    772
    773 302. The method of any one of claims 285 to 301, wherein said polymer backbone is
    774 poly(ethylene glycol).
    775
    776 303. The method of claim 302, wherein said poly(ethylene glycol) has an average
    777 molecular weight from about 500 Da to about 100,000 Da.
    778
    779 304. The method of any one of claims 285 to 303, wherein said preparation comprises
    780 at least 50% by weight of said water-soluble, non-peptidic, and non-nucleotidic
    781 polymer backbone having at least one terminus covalently bonded to a structure of
    782 formula (1).
    783
    784 305. The method of claim 304, wherein said preparation comprises at least 98% by
    785 weight of said water-soluble, non-peptidic, and non-nucleotidic polymer backbone
    786 having at least one terminus covalently bonded to a structure of formula (1).
    787
    788 306. The method of any one of claims 285 to 305, said method further comprising (i)
    789 removing the hydrophobic separation handle(s) from said structure of formula (1) and
    790 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    791 before reacting with a polypeptide that boosts red or white cell production or said
    792 derivative.
    793
    794 307. The method of any one of claims 285 to 306, said method further comprising
    795 removing the hydrophobic separation handle(s) from said structure of formula (1)
    796 after reacting with a polypeptide that boosts red or white cell production or said
    797 derivative. 798
    799 308. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a water-
    800 soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one
    801 terminus covalently bonded to a structure of formula (9):
    R1
    A O 1 L L2 B
    802
    803 or a salt thereof,
    804 wherein:
    805 A is the point of covalent bonding to the terminus of the polymer backbone;
    806 E is O or S;
    807 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    808 oligomeric alkyleneoxyalkylene;
    809 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic 81 o separation handle;
    81 1 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    812 can be NH;
    813 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    814 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    815 substituted arylene;
    816 R1 is absent or a hydrophobic separation handle, wherein only one of R1 and G can be
    817 a hydrophobic separation handle;
    818 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    819 B is a polypeptide that boosts red or white cell production or a derivative thereof. 820
    821 309. A method of making a polypeptide that boosts red or white cell production
    822 conjugate, said method comprising reacting a polypeptide that boosts red or white cell 823 production or a derivative thereof with a preparation comprising a compound of 824 formula (2) under conditions suitable for group M or R to react with a polypeptide 825 that boosts red or white cell production or said derivative thereof:
    R1 1 L1 Z3 P O polymer- -O- -M R
    Z4
    -K1 \
    826 (2)
    827 or a salt form thereof,
    828 wherein:
    829 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    830 backbone, wherein each linking group is bonded at a different terminus of said
    831 polymer;
    832 E and E1 are independently O or S;
    833 K and Ki are independently selected from the group consisting of: alkylene,
    834 alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    835 G and Gi are independently absent or are selected from the group consisting of:
    836 alkoxy and a hydrophobic separation handle;
    837 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    838 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    839 L and L1 are independently selected from the group consisting of: a divalent radical of
    840 a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and
    841 unsubstituted and substituted arylene;
    842 M and M1 are independently selected from a protected group that when deprotected is
    843 reactive with a polypeptide that boosts red or white cell production or said
    844 derivative or a group reactive with a polypeptide that boosts red or white cell
    845 production or said derivative, wherein M and M1 are different; and 846 R and R1 are independently absent, hydrogen, a protecting group, or an activating
    847 group;
    848 wherein when M is a protected group that when deprotected is reactive with a
    849 polypeptide that boosts red or white cell production or said derivative, then R is a
    850 protecting group or a hydrophobic separation handle;
    851 wherein when M is a group reactive with a polypeptide that boosts red or white cell
    852 production or said derivative, R is absent, hydrogen, or an activating group;
    853 wherein when M1 is a protected group that when deprotected is reactive with a
    854 polypeptide that boosts red or white cell production or said derivative, then R1 is a
    855 protecting group or a hydrophobic separation handle; and
    856 wherein when M1 is a group reactive with a polypeptide that boosts red or white cell
    857 production or said derivative, R1 is absent, hydrogen, or an activating group. 858
    859 310. The method of claim 309, wherein one of Z1 and Z2 is NH and the other is O. 860
    861 311. The method of claim 310, wherein Z1 is O and Z2 is NH.
    862
    863 312. The method of claim 310, wherein Z1 is NH and Z2 is O.
    864
    865 313. The method of claim 309, wherein Z1 is O and Z2 is O.
    866
    867 314. The method of claim 309, wherein one of Z3 and Z4 is NH and the other is O. 868
    869 315. The method of claim 314, wherein Z3 is O and Z4 is NH.
    870
    871 316. The method of claim 314, wherein Z3 is NH and Z4 is O.
    872
    873 317. The method of claim 309, wherein Z3 is O and Z4 is O.
    874
    875 318. The method of any one of claims 309 to 317, wherein said group reactive with a
    876 polypeptide that boosts red or white cell production or said derivative thereof is
    877 selected from the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde,
    878 glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,
    879 hydrazide, aminoxy, maleimide, dithiopyridine, iodoacetamide.
    880
    881 319. The method of any one of claims 309 to 318, wherein K or K1 is independently
    882 selected from the group consisting of: methylene, ethylene, propylene, isopropylene,
    883 butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from
    884 diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 885
    886 320. The method of any one of claims 309 to 319, wherein G or G1 is independently a
    887 substituted or unsubstituted trityloxy.
    888
    889 321. The method of any one of claims 309 to 320, wherein L or L1 is independently a
    890 substituted or unsubstituted alkylene.
    891
    892 322. The method of any one of claims 309 to 321, wherein R or R1 is independently
    893 selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    894 alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and
    895 combinations of thereof.
    896
    897 323. The method of any one of claims 309 to 322, wherein said polymer is
    898 poly(ethylene glycol).
    899
    900 324. The method of claim 323, wherein said poly(ethylene glycol) has an average
    901 molecular weight from about 500 Da to about 100,000 Da.
    902
    903 325. The method of any one of claims 309 to 324, wherein said preparation comprises
    904 at least 50% by weight of said compound. 905
    906 326. The method of any one of claims 309 to 325, said method further comprising (i)
    907 removing the hydrophobic separation handle(s) from said structure of formula (2) and
    908 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    909 before reacting with a polypeptide that boosts red or white cell production or said
    910 derivative.
    91 1
    912 327. The method of any one of claims 309 to 325, said method further comprising
    913 removing the hydrophobic separation handle(s) from said structure of formula (2)
    914 after reacting with a polypeptide that boosts red or white cell production or said
    915 derivative.
    916
    917 328. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a structure
    918 of formula (10):
    E1 E B1 L3 L1 Z3 P O polymer O P Z1 L L2 B
    Z4 Z2
    G 11 K1 \ K G
    919 (10)
    920 or a salt form thereof,
    921 wherein:
    922 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic
    923 polymer backbone, wherein each linking group is bonded at a different terminus
    924 of said polymer;
    925 E and E1 are independently O or S;
    926 K and Ki are independently selected from the group consisting of:
    927 alkylene, alkyleneoxyalkylene, and oligomeric alky leneoxy alky lene; 928 G and Gi are independently absent or are selected from the group
    929 consisting of: alkoxy and a hydrophobic separation handle;
    930 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O
    931 and NH, wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    932 L and L1 are independently selected from the group consisting of: a
    933 divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric
    934 alkyleneoxyalkylene, and unsubstituted and substituted arylene;
    935 L2 is a covalent linking moiety between L on the polymer backbone and
    936 B;
    937 L3 is a covalent linking moiety between L on the polymer backbone and
    938 B1; and
    939 B and B1 are independently a polypeptide that boosts red or white cell
    940 production, a derivative of a polypeptide that boosts red or white cell production,
    941 a biologic other than a polypeptide that boosts red or white cell production, a
    942 drug, a detectable group, a separation moiety, wherein at least one of B and B1 is
    943 a polypeptide that boosts red or white cell production or a derivative of a
    944 polypeptide that boosts red or white cell production.
    945
    946 329. A method of making a polypeptide that boosts red or white cell production
    947 conjugate, said method comprising reacting a polypeptide that boosts red or white cell
    948 production or a derivative thereof with a preparation comprising a compound of
    949 formula (3):
    E
    R2 M2 polymer O P Z L M R
    950 K G (3)
    951 or a salt form thereof,
    952 wherein: 953 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    954 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    955 at a different terminus of said polymer;
    956 E and E1 are independently O or S;
    957 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    958 oligomeric alkyleneoxyalkylene;
    959 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    960 separation handle;
    961 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    962 can be NH;
    963 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    964 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    965 substituted arylene;
    966 M is selected from a protected group that when deprotected is reactive with a
    967 polypeptide that boosts red or white cell production or said derivative or a group
    968 reactive with a polypeptide that boosts red or white cell production or said
    969 derivative;
    970 M2 is selected from O, S or NH; and
    971 R is absent, a protecting group, a hydrophobic separation handle, or an activating
    972 group;
    973 R2 is hydrogen or a protecting group;
    974 wherein when M is a protected group that when deprotected is reactive with a
    975 polypeptide that boosts red or white cell production or said derivative, then R is a
    976 protecting group or a hydrophobic separation handle; and
    977 wherein when M is a group reactive with a polypeptide that boosts red or white cell
    978 production or said derivative, R is absent, hydrogen, or an activating group.
    979
    980 330. The method of claim 329, wherein one of Z1 and Z2 is NH and the other is O. 981
    982 331. The method of claim 329 or claim 330, wherein G is a substituted or
    983 unsubstituted trityloxy group.
    984
    985 332. The method of claim 329, wherein G is selected from a monoalkoxy substituted
    986 trityloxy group or dialkoxy substituted trityloxy group.
    987
    988 333. The method of any one of claims 329 to 332, wherein said group reactive with a
    989 polypeptide that boosts red or white cell production or said derivative is selected from
    990 the group consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione,
    991 alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy,
    992 maleimide, dithiopyridine, iodoacetamide.
    993
    994 334. The method of any one of claims 329 to 333, wherein R2 is absent or selected
    995 from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    996 alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl,
    997 methyl.
    998
    999 335. The method of any one of claims 329 to 334, wherein said polymer is
    :000 poly(ethylene glycol).
    :ooi
    :002 336. The method of any one of claims 329 to 335, wherein said preparation comprises
    :003 at least 50% of said compound.
    :004
    :005 337. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a
    :006 compound of formula (11):
    B1 L4
    :007 (11) :008 or a salt form thereof,
    :009 wherein:
    :010 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    :01 1 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    :012 at a different terminus of said polymer;
    :013 E and E1 are independently O or S;
    :014 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and :015 oligomeric alkyleneoxyalkylene;
    :016 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic :o 17 separation handle;
    :018 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 :019 can be NH;
    :020 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene, :021 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :022 substituted arylene;
    :023 L2 is a covalent linking moiety between L on the polymer backbone and B;
    :024 L4 is a covalent linking moiety between L on the polymer backbone and B1; and
    :025 B and B1 are independently a polypeptide that boosts red or white cell production, a :026 derivative of a polypeptide that boosts red or white cell production, a biologic
    :027 other than a polypeptide that boosts red or white cell production, a drug, a
    :028 detectable group, a separation moiety, wherein at least one of B and B1 is a
    :029 polypeptide that boosts red or white cell production or a derivative of a
    :030 polypeptide that boosts red or white cell production.
    :031
    :032 338. A method of preparing a compound comprising a water-soluble, non-peptidic, and
    :033 non-nucleotidic polymer backbone having at least one terminus covalently bonded to
    :034 a structure of formula (9):
    :035 E R
    A O P Z L L2 B
    :036 Y (9)
    :037 or a salt thereof,
    :038 wherein:
    :039 A is the point of covalent bonding to the terminus of the polymer backbone;
    :040 E is O or S;
    :041 Y represents an optionally substituted residue selected from alkyl, cycloalkyl, :042 heterocyclyl, aryl, and heteroaryl;
    :043 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 :044 can be NH;
    :045 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, :046 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :047 substituted arylene;
    :048 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    :049 B is a polypeptide that boosts red or white cell production or a derivative thereof;
    :050 comprising the steps of:
    :051 (a) providing a composition comprising a compound of formula (8):
    :052
    :054 wherein:
    :055 A is the point of covalent bonding to the terminus of the polymer backbone;
    :056 E is O or S;
    :057 Y represents an optionally substituted residue selected from alkyl, cycloalkyl, :058 heterocyclyl, aryl, and heteroaryl; :059 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    :060 can be NH;
    :061 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene,
    :062 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :063 substituted arylene;
    :064 M is a protected group that when deprotected is reactive with a polypeptide that
    :065 boosts red or white cell production or a derivative thereof;
    :066 R is a hydrophobic separation handle;
    :067 R1 is absent or a hydrophobic separation handle; and
    :068 (b) removing the hydrophobic separation handle(s);
    :069 (c) optionally reacting the compound obtained in step (b) with an activating agent;
    :070 and
    :071 (d) reacting the compound obtained in step (b), or, optionally in step (c), with a
    :072 polypeptide that boosts red or white cell production or a derivative thereof.
    :073
    :074 339. The method of claim 338, wherein Z1 is O and Z2 is NH.
    :075
    :076 340. The method of claim 338, wherein Z1 is NH and Z2 is O.
    :077
    :078 341. The method of claim 338, wherein both Z1 and Z2 are O.
    :079
    :080 342. The method of any one of claims 338 to 341, wherein the protected group M, :081 when deprotected, is represented by hydroxyl, amine, thiol, carboxyl, aldehyde,
    :082 glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,
    :083 hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide.
    :084
    :085 343. The method of any one of claims 338 to 342, wherein R is selected from the :086 group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    :087 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    :088 :089 344. The method of any one of claims 338 to 343, wherein said polymer backbone is :090 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    :091 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    :092 polyoxazoline, and copolymers.
    :093
    :094 345. The method of any one of claims 338 to 344, wherein said polymer backbone is
    :095 poly(ethylene glycol).
    :096
    :097 346. The method of any one of claims 338 to 345, wherein reaction step (d) is carried
    :098 out in the presence of water or a protic solvent.
    :099
    00 347. The method of any one of claims 338 to 346, wherein the compound of formula
    101 (8) comprises at least 98% by weight of said composition.
    102
    103 348. A composition comprising the conjugate of any one of claims 308, 328, and 337,
    104 and a pharmaceutically acceptable excipient.
    105
    106 349. The composition of claim 348, wherein the polymer backbone of said conjugate is
    107 poly(ethylene glycol).
    108
    109 350. The composition of claim 348 or claim 349, wherein B is a polypeptide that
    1 1 o boosts red or white cell production.
    1 1 1
    1 12 351. The composition of any one of claims 348 to 350, wherein B1 is a polypeptide that
    1 13 boosts red or white cell production.
    1 14
    1 15 352. A method of treating a patient diagnosed with anemia or neutropenia, said method
    1 16 comprising administering to said patient an effective amount of the conjugate of any
    1 17 one of claims 308, 328, or 337.
    1 18
    353. The method of claim 352, wherein the polymer backbone of said conjugate is poly(ethylene glycol). 354. The method of claim 352 or claim 353, wherein B is a polypeptide that boosts red or white cell production. 355. The method of any one of claims 352 to 354, wherein B1 is a polypeptide that boosts red or white cell production. 356. A method of making an antibody conjugate, said method comprising reacting an antibody or a derivative thereof with a preparation comprising a water-soluble, non- peptidic, and non-nucleotidic polymer backbone having at least one terminus covalently bonded to a structure of formula (1) under conditions suitable for group M or R to react with an antibody or said derivative thereof:
    or a salt thereof,
    wherein:
    A is the point of covalent bonding to the terminus of the polymer backbone; E is O or S;
    K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic separation handle; 143 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    144 Z2 can be NH;
    145 L is selected from the group consisting of: a divalent radical of a nucleoside,
    146 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    147 substituted arylene;
    148 M is a protected group that when deprotected is reactive with an antibody or
    149 derivative thereof or is a group reactive with an antibody or derivative thereof;
    150 R is absent or selected from the group consisting of: hydrogen, a protecting group,
    151 a hydrophobic separation handle, or an activating group;
    152 R1 is absent or a hydrophobic separation handle;
    153 wherein when M is a protected group that when deprotected is reactive with an
    154 antibody or derivative thereof, then R is a protecting group or a hydrophobic separation
    155 handle;
    156 wherein when M is a group reactive with an antibody or derivative thereof, R is
    157 absent, hydrogen, or an activating group; and
    158 wherein only one of R, R1, and G can be a hydrophobic separation handle, .
    159
    160 357. The method of claim 356, wherein the polymer has from 2 to 100 termini.
    161
    162 358. The method of claim 356 or claim 357, wherein only one termini of the polymer
    163 backbone is covalently bonded to the structure of formula (1).
    164
    165 359. The method of any one of claims 356 to 358 wherein the polymer backbone has
    166 two termini.
    167
    168 360. The method of claim 359, wherein only one termini of the polymer backbone is
    169 covalently bonded to the structure of formula (1).
    170
    171 361. The method of claim 359, wherein both termini of the polymer backbone are
    172 covalently bonded to the structure of formula (1). 73
    174 362. The method of any one of claims 356 to 361, wherein one of Z1 and Z2 is NH
    175 and the other is O.
    176
    177 363. The method of claim 362, wherein Z1 is O and Z2 is NH.
    178
    179 364. The method of claim 362, wherein Z1 is NH and Z2 is O.
    180
    181 365. The method of claim 362, wherein both Z1 and Z2 are O.
    182
    183 366. The method of any one of claims 356 to 365, wherein said group reactive with an
    184 antibody or said derivative is selected from the group consisting of: hydroxyl, amine,
    185 thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    186 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    187 iodoacetamide.
    188
    189 367. The method of any one of claims 356 to 366, wherein K is selected from the
    190 group consisting of: methylene, ethylene, propylene, isopropylene, butylene,
    191 isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene
    192 glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    193
    194 368. The method of any one of claims 356 to 367 wherein G is a substituted or
    195 unsubstituted trityloxy.
    196
    197 369. The method of any one of claims 356 to 368, wherein L is a substituted or
    198 unsubstituted alkylene.
    199
    :200 370. The method any one of claims 356 to 369, wherein R is selected from the group
    201 consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    :202 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal. 203
    204 371. The method of any one of claims 356 to 370, wherein said group reactive with an
    205 antibody or said derivative is carboxyl and R is absent or selected from the group :206 consisting of: N-hydroxysuccinimidyl, p-nitrophenyl, or pentachlorophenyl.
    :207
    :208 372. The method of any one of claims 356 to 371, wherein said polymer backbone is
    209 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    210 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly( vinyl alcohol),
    21 1 polyoxazoline, and copolymers.
    212
    213 373. The method of any one of claims 356 to 372, wherein said polymer backbone is
    214 poly(ethylene glycol).
    215
    216 374. The method of claim 373, wherein said poly(ethylene glycol) has an average
    217 molecular weight from about 500 Da to about 100,000 Da.
    218
    219 375. The method of any one of claims 356 tp 374, wherein said preparation comprises
    220 at least 50% by weight of said water-soluble, non-peptidic, and non-nucleotidic
    221 polymer backbone having at least one terminus covalently bonded to a structure of
    222 formula (1).
    223
    224 376. The method of claim 375, wherein said preparation comprises at least 98% by
    225 weight of said water-soluble, non-peptidic, and non-nucleotidic polymer backbone
    226 having at least one terminus covalently bonded to a structure of formula (1).
    227
    228 377. The method of any one of claims 356 to 376, said method further comprising (i)
    229 removing the hydrophobic separation handle(s) from said structure of formula (1) and
    230 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    231 before reacting with an antibody or said derivative.
    232
    233 378. The method of any one of claims 356 to 377, said method further comprising
    234 removing the hydrophobic separation handle(s) from said structure of formula (1) :235 after reacting with an antibody or said derivative.
    :236
    :237 379. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a water- :238 soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one
    :239 terminus covalently bonded to a structure of formula (9):
    R
    A O 1 L L2 B
    :240
    241 or a salt thereof,
    :242 wherein:
    :243 A is the point of covalent bonding to the terminus of the polymer backbone;
    :244 E is O or S;
    :245 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    :246 oligomeric alkyleneoxyalkylene;
    :247 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    :248 separation handle;
    :249 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    :250 Z2 can be NH;
    251 L is selected from the group consisting of: a divalent radical of a nucleoside,
    252 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    253 substituted arylene;
    254 R1 is absent or a hydrophobic separation handle, wherein only one of R1 and G
    255 can be a hydrophobic separation handle;
    256 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    257 B is an antibody or a derivative thereof. 258
    259 380. A method of making an antibody conjugate, said method comprising reacting an 260 antibody or a derivative thereof with a preparation comprising a compound of 261 formula (2) under conditions suitable for group M or R to react with an antibody or !262 said derivative thereof:
    R1 1 L1 Z3 P O polymer- -O- -M R
    ,Z4
    -K1 \ K-
    !263
    !264 (2)
    :265 or a salt form thereof,
    :266 wherein:
    :267 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    :268 backbone, wherein each linking group is bonded at a different terminus of said polymer; :269 E and E1 are independently O or S;
    :270 K and Ki are independently selected from the group consisting of: alkylene,
    271 alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    272 G and Gi are independently absent or are selected from the group consisting of:
    273 alkoxy and a hydrophobic separation handle;
    274 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    275 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    276 L and L1 are independently selected from the group consisting of: a divalent
    277 radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene,
    278 and unsubstituted and substituted arylene;
    279 M and M1 are independently selected from a protected group that when
    280 deprotected is reactive with an antibody or said derivative or a group reactive with an
    281 antibody or said derivative, wherein M and M1 are different; and :282 R and R1 are independently absent, hydrogen, a protecting group, or an activating
    :283 group;
    :284 wherein when M is a protected group that when deprotected is reactive with an
    :285 antibody or said derivative, then R is a protecting group or a hydrophobic separation :286 handle;
    :287 wherein when M is a group reactive with an antibody or said derivative, R is
    :288 absent, hydrogen, or an activating group;
    :289 wherein when M1 is a protected group that when deprotected is reactive with an
    :290 antibody or said derivative, then R1 is a protecting group or a hydrophobic separation 291 handle; and
    :292 wherein when M1 is a group reactive with an antibody or said derivative, R1 is
    :293 absent, hydrogen, or an activating group.
    !294
    :295 381. The method of claim 380, wherein one of Z1 and Z2 is NH and the other is O. :296
    :297 382. The method of claim 381, wherein Z1 is O and Z2 is NH.
    !298
    :299 383. The method of claim 381, wherein Z1 is NH and Z2 is O.
    :300
    :301 384. The method of claim 380, wherein Z1 is O and Z2 is O.
    :302
    :303 385. The method of claim 380, wherein one of Z3 and Z4 is NH and the other is O.
    :304
    :305 386. The method of claim 385, wherein Z3 is O and Z4 is NH.
    :306
    :307 387. The method of claim 385, wherein Z3 is NH and Z4 is O.
    :308
    :309 388. The method of claim 380, wherein Z3 is O and Z4 is O.
    :310 :31 1 389. The method of any one of claims 380 to 388, wherein said group reactive with an :312 antibody or said derivative thereof is selected from the group consisting of: hydroxyl,
    :313 amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    :314 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    :315 iodoacetamide.
    :316
    :317 390. The method of any one of claims 380 to 389, wherein K or K1 is independently :318 selected from the group consisting of: methylene, ethylene, propylene, isopropylene,
    :319 butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from
    :320 diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    :321
    :322 391. The method of any one of claims 380 to 390, wherein G or G1 is independently a
    :323 substituted or unsubstituted trityloxy.
    !324
    :325 392. The method of any one of claims 380 to 391, wherein L or L1 is independently a
    :326 substituted or unsubstituted alkylene.
    !327
    :328 393. The method of any one of claims 380 to 392, wherein R or R1 is independently :329 selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    :330 alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and
    :331 combinations of thereof.
    !332
    :333 394. The method of any one of claims 380 to 393, wherein said polymer is
    :334 poly(ethylene glycol).
    !335
    :336 395. The method of claim 394, wherein said poly(ethylene glycol) has an average :337 molecular weight from about 500 Da to about 100,000 Da.
    !338
    :339 396. The method of any one of claims 380 to 395, wherein said preparation comprises :340 at least 50% by weight of said compound. 341
    :342 397. The method of any one of claims 380 to 396, said method further comprising (i) :343 removing the hydrophobic separation handle(s) from said structure of formula (2) and
    :344 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    :345 before reacting with an antibody or said derivative.
    !346
    :347 398. The method of any one of claims 380 to 396, said method further comprising :348 removing the hydrophobic separation handle(s) from said structure of formula (2)
    :349 after reacting with an antibody or said derivative.
    :350
    :351 399. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a structure :352 of formula (10):
    E1 E B1 L3 L1 Z3 P O polymer O P Z1 L L2 B
    Z4 Z2
    G 11 K /1 \ K G
    !353
    !354 (10)
    :355 or a salt form thereof,
    :356 wherein:
    :357 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    :358 backbone, wherein each linking group is bonded at a different terminus of said polymer; :359 E and E1 are independently O or S;
    :360 K and Ki are independently selected from the group consisting of: alkylene,
    :361 alky leneoxy alkylene, and oligomeric alkyleneoxyalkylene;
    :362 G and Gi are independently absent or are selected from the group consisting of:
    :363 alkoxy and a hydrophobic separation handle;
    :364 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    :365 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH; :366 L and L1 are independently selected from the group consisting of: a divalent
    :367 radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene,
    :368 and unsubstituted and substituted arylene;
    :369 L2 is a covalent linking moiety between L on the polymer backbone and B;
    :370 L3 is a covalent linking moiety between L on the polymer backbone and B1; and
    :371 B and B1 are independently an antibody, a derivative of an antibody, a biologic
    :372 other than an antibody, a drug, a detectable group, a separation moiety, wherein at least
    :373 one of B and B1 is an antibody or a derivative of an antibody.
    !374
    :375 400. A method of making an antibody conjugate, said method comprising reacting an :376 antibody or a derivative thereof with a preparation comprising a compound of
    :377 formula (3):
    E
    R2 M2 polymer O P Z L M R
    :378 K G (3)
    :379 or a salt form thereof,
    :380 wherein:
    :381 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    :382 backbone, wherein M2 and the phosphonate-derived functional group are bonded at a
    :383 different terminus of said polymer;
    :384 E and E1 are independently O or S;
    :385 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    :386 oligomeric alkyleneoxyalkylene;
    :387 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    :388 separation handle;
    :389 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    :390 Z2 can be NH; :391 L is selected from the group consisting of: a divalent radical of nucleoside,
    :392 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :393 substituted arylene;
    :394 M is selected from a protected group that when deprotected is reactive with an
    :395 antibody or said derivative or a group reactive with an antibody or said derivative;
    :396 M2 is selected from O, S or NH; and
    :397 R is absent, a protecting group, a hydrophobic separation handle, or an activating
    :398 group;
    :399 R2 is hydrogen or a protecting group;
    :400 wherein when M is a protected group that when deprotected is reactive with an
    :401 antibody or said derivative, then R is a protecting group or a hydrophobic separation
    :402 handle; and
    :403 wherein when M is a group reactive with an antibody or said derivative, R is
    :404 absent, hydrogen, or an activating group.
    :405
    :406 401. The method of claim 400 wherein one of Z1 and Z2 is NH and the other is O. :407
    :408 402. The method of claim 400 or claim 401, wherein G is a substituted or
    :409 unsubstituted trityloxy group.
    :410
    :41 1 403. The method of claim 400, wherein G is selected from a monoalkoxy substituted
    :412 trityloxy group or dialkoxy substituted trityloxy group.
    :413
    :414 404. The method of any one of claims 400 to 403, wherein said group reactive with an
    :415 antibody or said derivative is selected from the group consisting of: hydroxyl, amine,
    :416 thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide,
    :417 acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide, dithiopyridine,
    :418 iodoacetamide .
    :419
    :420 :421 405. The method of any one of claims 400 to 404, wherein R2 is absent or selected
    :422 from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    :423 alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl,
    :424 methyl.
    !425
    :426 406. The method of any one of claims 400 to 405, wherein said polymer is
    :427 poly(ethylene glycol).
    !428
    :429 407. The method of any one of claims 400 to 406, wherein said preparation comprises
    :430 at least 50% of said compound.
    :431
    :432 408. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a
    :433 compound of formula (11):
    :435 or a salt form thereof,
    :436 wherein:
    :437 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    :438 backbone, wherein M2 and the phosphonate-derived functional group are bonded at a
    :439 different terminus of said polymer;
    :440 E and E1 are independently O or S;
    :441 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    :442 oligomeric alkyleneoxyalkylene;
    :443 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    :444 separation handle;
    :445 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    :446 Z2 can be NH; :447 L is selected from the group consisting of: a divalent radical of nucleoside,
    :448 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :449 substituted arylene;
    :450 L2 is a covalent linking moiety between L on the polymer backbone and B;
    :451 L4 is a covalent linking moiety between L on the polymer backbone and B1; and
    :452 B and B1 are independently an antibody, a derivative of an antibody, a biologic
    :453 other than an antibody, a drug, a detectable group, a separation moiety, wherein at least
    :454 one of B and B1 is an antibody or a derivative of an antibody.
    !455
    :456 409. A method of preparing a compound comprising a water-soluble, non-peptidic, :457 and non-nucleotidic polymer backbone having at least one terminus covalently
    :458 bonded to a structure of formula (9):
    !459
    E R A O P Z L L2 B
    :460 Y (9)
    :461 or a salt thereof,
    :462 wherein:
    :463 A is the point of covalent bonding to the terminus of the polymer backbone;
    :464 E is O or S;
    :465 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    :466 heterocyclyl, aryl, and heteroaryl;
    :467 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    :468 Z2 can be NH;
    :469 L is selected from the group consisting of: a divalent radical of a nucleoside,
    :470 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :471 substituted arylene;
    :472 L2 is a covalent linking moiety between L on the polymer backbone and B; and :473 B is an antibody or a derivative thereof;
    :474 comprising the steps of:
    :475 (a) providing a composition comprising a compound of formula (8):
    !476
    :478 wherein:
    :479 A is the point of covalent bonding to the terminus of the polymer backbone;
    :480 E is O or S;
    :481 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    :482 heterocyclyl, aryl, and heteroaryl;
    :483 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and
    :484 Z2 can be NH;
    :485 L is selected from the group consisting of: a divalent radical of a nucleoside,
    :486 alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :487 substituted arylene;
    :488 M is a protected group that when deprotected is reactive with an antibody or a
    :489 derivative thereof;
    :490 R is a hydrophobic separation handle;
    :491 R1 is absent or a hydrophobic separation handle; and
    :492 (b) removing the hydrophobic separation handle(s);
    :493 (c) optionally reacting the compound obtained in step (b) with an activating agent;
    :494 and
    :495 (d) reacting the compound obtained in step (b), or, optionally in step (c), with an
    :496 antibody or a derivative thereof.
    !497
    :498 410. The method of claim 409, wherein Z1 is O and Z2 is NH. !499
    :500 411. The method of claim 409, wherein Z1 is NH and Z2 is O.
    :501
    :502 412. The method of claim 409, wherein both Z1 and Z2 are O.
    :503
    :504 413. The method of any one of claims 409 to 412, wherein the protected group M, :505 when deprotected, is represented by hydroxyl, amine, thiol, carboxyl, aldehyde,
    :506 glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,
    :507 hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide.
    :508
    :509 414. The method of any one of claims 409 to 413, wherein R is selected from the :510 group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    :51 1 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    :512
    :513 415. The method of any one of claims 409 to 414, wherein said polymer backbone is :514 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    :515 polyol), poly(olefinic alcohol), poly(a-hydroxy acid), poly( vinyl alcohol),
    :516 polyoxazoline, and copolymers.
    :517
    :518 416. The method of any one of claims 409 to 415, wherein said polymer backbone is
    :519 poly(ethylene glycol).
    :520
    :521 417. The method of any one of claims 409 to 416, wherein reaction step (d) is carried
    :522 out in the presence of water or a protic solvent.
    !523
    :524 418. The method of any one of claims 409 to 417, wherein the compound of formula
    :525 (8) comprises at least 98% by weight of said composition.
    !526
    :527 419. A composition comprising the conjugate of any one of claims 379, 399, and 408, :528 and a pharmaceutically acceptable excipient. !529
    :530 420. The composition of claim 419, wherein the polymer backbone of said conjugate
    :531 is poly(ethylene glycol).
    !532
    :533 421. The composition of claim 419 or claim 420, wherein B is an antibody.
    !534
    :535 422. The composition of any one of claims 419 to 421, wherein B1 is an antibody. !536
    :537 423. A method of making a biologically active molecule conjugate, said method :538 comprising reacting a biologically active molecule or a derivative thereof with a
    :539 preparation comprising a water-soluble, non-peptidic, and non-nucleotidic polymer
    :540 backbone having at least one terminus covalently bonded to a structure of formula (1)
    :541 under conditions suitable for group M or R to react with a biologically active
    :542 molecule or said derivative thereof:
    !543
    :545 or a salt thereof,
    :546 wherein:
    :547 A is the point of covalent bonding to the terminus of the polymer backbone;
    :548 E is O or S;
    :549 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and :550 oligomeric alkyleneoxyalkylene;
    :551 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic :552 separation handle; :553 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 :554 can be NH;
    :555 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, :556 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :557 substituted arylene;
    :558 M is a protected group that when deprotected is reactive with a biologically active :559 molecule or derivative thereof or is a group reactive with a biologically active
    :560 molecule or derivative thereof;
    :561 R is absent or selected from the group consisting of: hydrogen, a protecting group, a :562 hydrophobic separation handle, or an activating group;
    :563 R1 is absent or a hydrophobic separation handle;
    :564 wherein when M is a protected group that when deprotected is reactive with a
    :565 biologically active molecule or derivative thereof, then R is a protecting group or
    :566 a hydrophobic separation handle;
    :567 wherein when M is a group reactive with a biologically active molecule or derivative
    :568 thereof, R is absent, hydrogen, or an activating group; and
    :569 wherein only one of R, R1, and G can be a hydrophobic separation handle, .
    :570
    :571 424. The method of claim 423, wherein the polymer has from 2 to 100 termini.
    !572
    :573 425. The method of claim 423 or claim 424, wherein only one termini of the polymer
    :574 backbone is covalently bonded to the structure of formula (1).
    !575
    :576 426. The method of any one of claims 423 to 425 wherein the polymer backbone has
    :577 two termini.
    !578
    :579 427. The method of claim 426, wherein only one termini of the polymer backbone is
    :580 covalently bonded to the structure of formula (1).
    :581 :582 428. The method of claim 426, wherein both termini of the polymer backbone are
    :583 covalently bonded to the structure of formula (1).
    !584
    1585 429. The method of any one of claims 423 to 428, wherein one of Z1 and Z2 is NH and :586 the other is O.
    !587
    :588 430. The method of claim 429, wherein Z1 is O and Z2 is NH.
    !589
    :590 431. The method of claim 429, wherein Z1 is NH and Z2 is O.
    :591
    '592 432. The method of claim 429, wherein both Z1 and Z2 are O.
    !593
    :594 433. The method of any one of claims 423 to 432, wherein said group reactive with a :595 biologically active molecule or said derivative is selected from the group consisting
    :596 of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl,
    :597 alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,
    :598 dithiopyridine, iodoacetamide.
    !599
    :600 434. The method of any one of claims 423 to 433, wherein K is selected from the
    :601 group consisting of: methylene, ethylene, propylene, isopropylene, butylene,
    :602 isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from diethylene
    :603 glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol.
    :604
    :605 435. The method of any one of claims 423 to 434, wherein G is a substituted or
    :606 unsubstituted trityloxy.
    :607
    :608 436. The method of any one of claims 423 to 435, wherein L is a substituted or
    :609 unsubstituted alkylene.
    :610 :61 1 437. The method any one of claims 423 to 436, wherein R is selected from the group :612 consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    :613 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    :614
    :615 438. The method of any one of claims 423 to 437, wherein said group reactive with a :616 biologically active molecule or said derivative is carboxyl and R is absent or selected
    :617 from the group consisting of: N-hydroxysuccinimidyl, p-nitrophenyl, or
    :618 pentachlorophenyl.
    :619
    :620 439. The method of any one of claims 423 to 438, wherein said polymer backbone is :621 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    :622 polyol), poly(olefmic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    :623 polyoxazoline, and copolymers.
    !624
    :625 440. The method of any one of claims 423 to 439, wherein said polymer backbone is
    :626 poly(ethylene glycol).
    !627
    :628 441. The method of claim 440, wherein said poly(ethylene glycol) has an average :629 molecular weight from about 500 Da to about 100,000 Da.
    :630
    :631 442. The method of any one of claims 423 to 441, wherein said preparation comprises :632 at least 50% by weight of said water-soluble, non-peptidic, and non-nucleotidic
    :633 polymer backbone having at least one terminus covalently bonded to a structure of
    :634 formula (1).
    !635
    :636 443. The method of claim 442, wherein said preparation comprises at least 98% by :637 weight of said water-soluble, non-peptidic, and non-nucleotidic polymer backbone
    :638 having at least one terminus covalently bonded to a structure of formula (1).
    :639 :640 444. The method of any one of claims 423 to 443, said method further comprising (i) :641 removing the hydrophobic separation handle(s) from said structure of formula (1) and
    :642 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    :643 before reacting with a biologically active molecule or said derivative.
    !644
    :645 445. The method of any one of claims 423 to 443, said method further comprising :646 removing the hydrophobic separation handle(s) from said structure of formula (1)
    :647 after reacting with a biologically active molecule or said derivative.
    !648
    :649 446. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a water- :650 soluble, non-peptidic, and non-nucleotidic polymer backbone having at least one
    :651 terminus covalently bonded to a structure of formula (9):
    R1
    A O 1 L L2 B
    :652
    :653 or a salt thereof,
    :654 wherein:
    :655 A is the point of covalent bonding to the terminus of the polymer backbone;
    :656 E is O or S;
    :657 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and :658 oligomeric alkyleneoxyalkylene;
    :659 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic :660 separation handle;
    :661 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 :662 can be NH; :663 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, :664 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :665 substituted arylene;
    :666 R1 is absent or a hydrophobic separation handle, wherein only one of R1 and G can be :667 a hydrophobic separation handle;
    :668 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    :669 B is a biologically active molecule or a derivative thereof.
    :670
    :671 447. A method of making a biologically active molecule conjugate, said method
    :672 comprising reacting a biologically active molecule or a derivative thereof with a
    :673 preparation comprising a compound of formula (2) under conditions suitable for
    :674 group M or R to react with a biologically active molecule or said derivative thereof:
    R1 1 L1 Z3 P 11 O polymer O P \\ Z1 L M R
    G1 K1 /' K G
    !675 (2)
    :676 or a salt form thereof,
    :677 wherein:
    :678 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    :679 backbone, wherein each linking group is bonded at a different terminus of said
    :680 polymer;
    :681 E and E1 are independently O or S;
    :682 K and Ki are independently selected from the group consisting of: alkylene, :683 alkyleneoxyalkylene, and oligomeric alkyleneoxyalkylene;
    :684 G and Gi are independently absent or are selected from the group consisting of: :685 alkoxy and a hydrophobic separation handle; :686 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O and NH,
    :687 wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    :688 L and L1 are independently selected from the group consisting of: a divalent radical of
    :689 a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and
    :690 unsubstituted and substituted arylene;
    :691 M and M1 are independently selected from a protected group that when deprotected is
    :692 reactive with a biologically active molecule or said derivative or a group reactive
    :693 with a biologically active molecule or said derivative, wherein M and M1 are
    :694 different; and
    :695 R and R1 are independently absent, hydrogen, a protecting group, or an activating
    :696 group;
    :697 wherein when M is a protected group that when deprotected is reactive with a
    :698 biologically active molecule or said derivative, then R is a protecting group or a
    :699 hydrophobic separation handle;
    700 wherein when M is a group reactive with a biologically active molecule or said
    701 derivative, R is absent, hydrogen, or an activating group;
    702 wherein when M1 is a protected group that when deprotected is reactive with a
    703 biologically active molecule or said derivative, then R1 is a protecting group or a
    704 hydrophobic separation handle; and
    705 wherein when M1 is a group reactive with a biologically active molecule or said
    706 derivative, R1 is absent, hydrogen, or an activating group.
    707
    708 448. The method of claim 447, wherein one of Z1 and Z2 is NH and the other is O. 709
    710 449. The method of claim 448, wherein Z1 is O and Z2 is NH.
    71 1
    712 450. The method of claim 448, wherein Z1 is NH and Z2 is O.
    713
    714 451. The method of claim 447, wherein Z1 is O and Z2 is O.
    715
    716 452. The method of claim 447, wherein one of Z3 and Z4 is NH and the other is O.
    717
    718 453. The method of claim 452, wherein Z3 is O and Z4 is NH.
    719
    720 454. The method of claim 452, wherein Z3 is NH and Z4 is O.
    721
    722 455. The method of claim 447, wherein Z3 is O and Z4 is O.
    723
    724 456. The method of any one of claims 447 to 455, wherein said group reactive with a
    725 biologically active molecule or said derivative thereof is selected from the group
    726 consisting of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl,
    727 alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,
    728 dithiopyridine, iodoacetamide.
    729
    730 457. The method of any one of claims 447 to 456, wherein K or K1 is independently
    731 selected from the group consisting of: methylene, ethylene, propylene, isopropylene,
    732 butylene, isobutylene, sec-butylene, tert-butylene, and hexylene, or a residue from
    733 diethylene glycol, triethylene glycol, tetraethylene glycol or hexaethylene glycol. 734
    735 458. The method of any one of claims 447 to 457, wherein G or G1 is independently a
    736 substituted or unsubstituted trityloxy.
    737
    738 459. The method of any one of claims 447 to 458, wherein L or L1 is independently a
    739 substituted or unsubstituted alkylene.
    740
    741 460. The method of any one of claims 447 to 459, wherein R or R1 is independently
    742 selected from the group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    743 alkoxypixyl, fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, cyclic acetal, and
    744 combinations of thereof.
    745
    746 461. The method of any one of claims 447 to 460, wherein said polymer is
    747 poly(ethylene glycol).
    748
    749 462. The method of claim 461, wherein said poly(ethylene glycol) has an average
    750 molecular weight from about 500 Da to about 100,000 Da.
    751
    752 463. The method of any one of claims 447 to 462, wherein said preparation comprises
    753 at least 50% by weight of said compound.
    754
    755 464. The method of any one of claims 447 to 463, said method further comprising (i)
    756 removing the hydrophobic separation handle(s) from said structure of formula (2) and
    757 (ii) optionally reacting the compound obtained in step (i) with an activating agent
    758 before reacting with a biologically active molecule or said derivative.
    759
    760 465. The method of any one of claims 447 to 463, said method further comprising
    761 removing the hydrophobic separation handle(s) from said structure of formula (2)
    762 after reacting with a biologically active molecule or said derivative.
    763
    764 466. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a structure
    765 of formula (10):
    E1 E B1 L3 L1 Z3 P O polymer O P Z1 L L2 B
    Z4 Z2
    G 11 K1 \ K G
    766 (10)
    767 or a salt form thereof,
    768 wherein: 769 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic
    770 polymer backbone, wherein each linking group is bonded at a different terminus
    771 of said polymer;
    772 E and E1 are independently O or S;
    773 K and Ki are independently selected from the group consisting of:
    774 alkylene, alkyleneoxyalkylene, and oligomeric alky leneoxy alky lene;
    775 G and Gi are independently absent or are selected from the group
    776 consisting of: alkoxy and a hydrophobic separation handle;
    777 each pair of Z1 and Z2 and Z3 and Z4 are independently selected from O
    778 and NH, wherein only one of each pair of Z1 and Z2 and Z3 and Z4 can be NH;
    779 L and L1 are independently selected from the group consisting of: a
    780 divalent radical of a nucleoside, alkylene, alkyleneoxyalkylene, oligomeric
    781 alkyleneoxyalkylene, and unsubstituted and substituted arylene;
    782 L2 is a covalent linking moiety between L on the polymer backbone and
    783 B;
    784 L3 is a covalent linking moiety between L on the polymer backbone and
    785 B1; and
    786 B and B1 are independently a biologically active molecule, a derivative of
    787 a biologically active molecule, a biologic other than a biologically active
    788 molecule, a drug, a detectable group, a separation moiety, wherein at least one of
    789 B and B1 is a biologically active molecule or a derivative of a biologically active
    790 molecule.
    791
    792 467. A method of making a biologically active molecule conjugate, said method
    793 comprising reacting a biologically active molecule or a derivative thereof with a
    794 preparation comprising a compound of formula (3): E
    R2 M2 polymer O P Z L M R
    795 K G (3)
    796 or a salt form thereof,
    797 wherein:
    798 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    799 backbone, wherein M2 and the phosphonate-derived functional group are bonded :800 at a different terminus of said polymer;
    :801 E and E1 are independently O or S;
    :802 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and :803 oligomeric alkyleneoxyalkylene;
    :804 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic :805 separation handle;
    :806 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 :807 can be NH;
    :808 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene, :809 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :810 substituted arylene;
    :81 1 M is selected from a protected group that when deprotected is reactive with a :812 biologically active molecule or said derivative or a group reactive with a
    :813 biologically active molecule or said derivative;
    :814 M2 is selected from O, S or NH; and
    :815 R is absent, a protecting group, a hydrophobic separation handle, or an activating :816 group;
    :817 R2 is hydrogen or a protecting group;
    :818 wherein when M is a protected group that when deprotected is reactive with a
    :819 biologically active molecule or said derivative, then R is a protecting group or a
    :820 hydrophobic separation handle; and :821 wherein when M is a group reactive with a biologically active molecule or said
    :822 derivative, R is absent, hydrogen, or an activating group.
    !823
    :824 468. The method of claim 467, wherein one of Z1 and Z2 is NH and the other is O. !825
    :826 469. The method of claim 467 or claim 468, wherein G is a substituted or
    :827 unsubstituted trityloxy group.
    !828
    :829 470. The method of claim 467, wherein G is selected from a monoalkoxy substituted
    :830 trityloxy group or dialkoxy substituted trityloxy group.
    :831
    :832 471. The method of any one of claims 467 to 470, wherein said group reactive with a :833 biologically active molecule or said derivative is selected from the group consisting
    :834 of: hydroxyl, amine, thiol, carboxyl, aldehyde, glyoxal, dione, alkenyl, alkynyl,
    :835 alkedienyl, azide, acrylamide, vinyl sulfone, hydrazide, aminoxy, maleimide,
    :836 dithiopyridine, iodoacetamide.
    !837
    :838 472. The method of any one of claims 467 to 471, wherein R2 is absent or selected
    :839 from the group consisting of trityl, monoalkoxytrityl, dialkoxytrityl, pixyl,
    :840 alkoxypixyl, fluorenylmethyloxycarbonyl, alkylcarboxyl, benzoyl, tetrahydropyranyl,
    :841 methyl.
    :842
    :843 473. The method of any one of claims 467 to 472, wherein said polymer is
    :844 poly(ethylene glycol).
    !845
    :846 474. The method of any one of claims 467 to 473, wherein said preparation comprises
    :847 at least 50% of said compound.
    !848
    :849 475. A conjugate, or a pharmaceutically acceptable salt thereof, comprising a
    :850 compound of formula (11): B1 L4 polymer O P Z1 L L2
    [2 B
    \
    1851 K G (11)
    :852 or a salt form thereof,
    :853 wherein:
    :854 polymer is a linear, water-soluble, non-peptidic, and non-nucleotidic polymer
    :855 backbone, wherein M2 and the phosphonate-derived functional group are bonded
    :856 at a different terminus of said polymer;
    :857 E and E1 are independently O or S;
    :858 K is selected from the group consisting of: alkylene, alkyleneoxyalkylene, and
    :859 oligomeric alkyleneoxyalkylene;
    :860 G is selected from the group consisting of: hydrogen, alkoxy, and a hydrophobic
    :861 separation handle;
    :862 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2
    :863 can be NH;
    :864 L is selected from the group consisting of: a divalent radical of nucleoside, alkylene,
    :865 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :866 substituted arylene;
    :867 L2 is a covalent linking moiety between L on the polymer backbone and B;
    :868 L4 is a covalent linking moiety between L on the polymer backbone and B1; and
    :869 B and B1 are independently a biologically active molecule, a derivative of a
    :870 biologically active molecule, a biologic other than a biologically active molecule,
    :871 a drug, a detectable group, a separation moiety, wherein at least one of B and B1 is
    :872 a biologically active molecule or a derivative of a biologically active molecule.
    !873
    :874 476. A method of preparing a compound comprising a water-soluble, non-peptidic, and
    :875 non-nucleotidic polymer backbone having at least one terminus covalently bonded to
    :876 a structure of formula (9): !877
    E R
    A O P Z L L2 B
    :878 Y (9)
    :879 or a salt thereof,
    :880 wherein:
    :881 A is the point of covalent bonding to the terminus of the polymer backbone;
    :882 E is O or S;
    :883 Y represents an optionally substituted residue selected from alkyl, cycloalkyl,
    :884 heterocyclyl, aryl, and heteroaryl;
    :885 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 :886 can be NH;
    :887 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, :888 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :889 substituted arylene;
    :890 L2 is a covalent linking moiety between L on the polymer backbone and B; and
    :891 B is a biologically active molecule or a derivative thereof;
    :892 comprising the steps of:
    :893 (a) providing a composition comprising a compound of formula (8):
    !894
    :896 wherein:
    :897 A is the point of covalent bonding to the terminus of the polymer backbone;
    :898 E is O or S; :899 Y represents an optionally substituted residue selected from alkyl, cycloalkyl, :900 heterocyclyl, aryl, and heteroaryl;
    :901 Z1 and Z2 are independently selected from O and NH, wherein only one of Z1 and Z2 :902 can be NH;
    :903 L is selected from the group consisting of: a divalent radical of a nucleoside, alkylene, :904 alkyleneoxyalkylene, oligomeric alkyleneoxyalkylene, and unsubstituted and
    :905 substituted arylene;
    :906 M is a protected group that when deprotected is reactive with a biologically active
    :907 molecule or a derivative thereof;
    :908 R is a hydrophobic separation handle;
    :909 R1 is absent or a hydrophobic separation handle; and
    :910 (b) removing the hydrophobic separation handle(s);
    :91 1 (c) optionally reacting the compound obtained in step (b) with an activating agent; :912 and
    :913 (d) reacting the compound obtained in step (b), or, optionally in step (c), with a
    :914 biologically active molecule or a derivative thereof.
    :915
    :916 477. The method of claim 476, wherein Z1 is O and Z2 is NH.
    :917
    :918 478. The method of claim 476, wherein Z1 is NH and Z2 is O.
    :919
    :920 479. The method of claim 476, wherein both Z1 and Z2 are O.
    :921
    :922 480. The method of any one of claims 476 to 479, wherein the protected group M,
    :923 when deprotected, is represented by hydroxyl, amine, thiol, carboxyl, aldehyde,
    :924 glyoxal, dione, alkenyl, alkynyl, alkedienyl, azide, acrylamide, vinyl sulfone,
    :925 hydrazide, aminoxy, maleimide, dithiopyridine, or iodoacetamide.
    :926 :927 481. The method of any one of claims 476 to 480, wherein R is selected from the :928 group consisting of: trityl, monoalkoxytrityl, dialkoxytrityl, pixyl, alkoxypixyl,
    :929 fluorenylmethyloxycarbonyl, trifluoroacetyl, acetal, and cyclic acetal.
    :930
    :931 482. The method of any one of claims 476 to 481 , wherein said polymer backbone is :932 selected from the group consisting of poly(alkylene glycol), poly(oxyethylated
    :933 polyol), poly(olefinic alcohol), poly(a-hydroxy acid), poly(vinyl alcohol),
    :934 polyoxazoline, and copolymers.
    !935
    :936 483. The method of any one of claims 476 to 482, wherein said polymer backbone is
    :937 poly(ethylene glycol).
    :938
    :939 484. The method of any one of claims 476 to 483, wherein reaction step (d) is carried
    :940 out in the presence of water or a protic solvent.
    :941
    !942
    !943
    1
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